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Biomedical subjects

T Fukao

Publications and source records attributed to T Fukao.

At least 19 recordsLinked to original sources
PubMed2006

Effect of Hunter disease (mucopolysaccharidosis type II) mutations on molecular phenotypes of iduronate-2-sulfatase: enzymatic activity, protein processing and structural analysis.

Mucopolysaccharidosis II (Hunter disease), a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), has variable clinical phenotypes. Nearly 300 different mutations have been identified in the IDS gene from patients with Hunter disease, but the correlation between the genotype and phenotype has remained unclear. We studied the characteristics of 11 missense mutations, which were detected in the patients or artificially introduced, using stable expression experiments and structural analysis. The mutants found in the attenuated phenotype showed considerable residual activity (0.2-2.4% of the wild-type IDS activity) and those in the severe phenotype had no activity. In immunoblot analysis, both the 73-75 kDa precursor and processed forms were detected in the expression of 'attenuated' mutants (R48P, A85T and W337R) and the artificial active site mutants (C84S, C84T). The 73-75 kDa initial precursor was detected in the 'severe' mutants (P86L, S333L, S349I, R468Q, R468L). The truncated 68 kDa precursor form was synthesized in the Q531X mutant. The results of immunoblotting indicated rapid degradation and/or insufficiency in processing as a result of structural alteration of the IDS protein. A combination of analyses of genotype and molecular phenotypes, including enzyme activity, protein processing and structural analysis with an engineered reference protein, could provide an avenue to understanding the molecular mechanism of the disease and could give a useful tool for the evaluation of possible therapeutic chemical compounds.

Animals↗
PubMed2005

Leaky phenotype of X-linked agammaglobulinaemia in a Japanese family.

X-linked agammaglobulinaemia (XLA) is an inherited immunodeficiency that is caused by a block in early B-cell differentiation. Whereas early B precursors in the bone marrow are present in substantial numbers, XLA-affected individuals have dramatically reduced numbers of circulating mature B cells, plasma cells and immunoglobulins of all isotypes. We report on a Japanese family with 3 XLA patients, in whom the serum immunoglobulin levels and number of B cells showed a significant difference among them in spite of harbouring the same splice donor site mutation in the BTK gene. We developed concise method for detection of this mutation, which is helpful for discovering the carrier. Patient 2 showed a significant serum immunoglobulin levels of all isotypes, including allergen-specific IgE. Expression of a normal and truncated size BTK gene was detected in patient 2's peripheral blood mononuclear cells (PBMCs). Expression of BTK protein was also detected in some B cells. These results suggest that the leaky phenotype in patient 2 was caused in part by the expression of a normal BTK gene transcript. The increased frequency of infection with age expanded the number of B cells with normal BTK gene expression and produced the serum immunoglobulin, including IgE.

Adult↗
PubMed2005

Characterization of T-cell clones specific to ovomucoid from patients with egg-white allergy.

BACKGROUND: Allergic reactions to foods are specific problems for infants and young children. Ovomucoid (OM) is one of the major allergens found in egg-white. We previously established several T-cell clones (TCCs) specific to OM in non-polarizing conditions from 4 patients (TM and YN are immediate-type, IH and YT are non-immediate-type) with egg-white allergy. We characterized their reactive epitopes, antigen-presenting molecules (HLA class II), and usage of TCR alpha and beta genes and the CDR3 loop sequence. OBJECTIVE: The objective of this study was to characterize these seven clones (TM 1.3, TM1.4,YN 1.1, YN1.5, IH3.1, IH3.3 and YT6.1) for cytokine production patterns and cell-surface-marker phenotypes. METHODS: We measured the production of cytokines, namely interleukin (IL)-4, IL-5 and interferon-gamma (IFN-gamma) by stimulation with ovomucoid peptides and stained intracellular IL-4 and IFN-gamma, and determined cell-surface markers using anti-interleukin-12 receptor (IL-12R) beta1, anti-IL-12Rbeta2 and anti-interleukin-18 receptor alpha (IL-18Ralpha). RESULTS: Most TCCs secreted both IL-4 and IFN-gamma in response to the OM peptide mixture, but the secretion patterns were variable; an IFN-gamma dominant pattern was seen in IH3.1 andYT6.1, an IFN-gamma>IL-4 pattern in TM1.3 and TM1.4, an IL-4> IFN-gamma pattern in YN1.5. In intracellular IFN-gamma and IL-4 staining, IFN-gamma single-positive cells were predominant in TM1.3, TM1.4, IH3.1 and YT6.1 and IFN-gamma and IL-4 double-positive cells were predominant in YN1.1, YN1.5 and IH3.3. All TCCs were IL-12Rbeta1-positive, and TM1.3, IH3.1, IH3.3 and YT6.1 were both IL-12Rbeta2- and IL-18Ralpha-positive. TM1.4 and YN1.1 were both IL-12Rbeta2- and IL-18Ralpha-negative. Based on these results, TM1.3 and TM1.4, IH3.1 and YT6.1 had a predominantly Th1 character and YN1.1, YN1.5, and IH3.3 possessed a predominantly Th0 character. CONCLUSIONS: The phenotypes of TCCs were not in accordance with their clinical manifestations. TCCs established from patients with immediate-type hypersensitivity had either the Th1 or Th0 phenotype as well as those with non-immediate-type hypersensitivity.

Egg Hypersensitivity↗
PubMed2004

The effects of allyl sulfides on the induction of phase II detoxification enzymes and liver injury by carbon tetrachloride.

Allyl sulfides, e.g., diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), are principal constituents of garlic oil. In the present study, we investigated the in vivo effect of these sulfides on the phase I and phase II drug-metabolizing enzymes, and elucidated their structure-function relationship. A highly purified form of each sulfide (more than 99% purity) was administered i.p. as a bolus to rats at a concentration of 10 or 100 micromol/kg body weight for 14 consecutive days. As to the phase I enzymes, DAS (100 micromol/kg) slightly but significantly increased cytochrome P-450 (CYP) 2E1 activity (1.6-fold vs. control), whereas DADS and DATS did not affect it or the hepatic total CYP level or CYP1A1/2 activity. With respect to the phase II enzymes, DATS (10 micromol/kg) and DADS at a 10-fold higher dose (100 micromol/kg) significantly increased the activities of glutathione S-transferase, quinone reductase, and antioxidative enzyme glutathione peroxidase; whereas DAS did not. In the carbon tetrachloride (CCl4)-induced acute liver injury model of rats, either DATS (10 micromol/kg) or DADS (100 micromol/kg) significantly reduced the injury caused by the induction of phase II enzymes with CCl4. In conclusion, the sulfides affected both phase I and phase II enzymes, the former being stimulated by the monosulfide only and the latter, strongly by the trisulfide and weakly by the disulfide. Therefore, the polysulfide DATS may be one of the important factors in garlic oil that protects our body against the injury caused by radical molecules encountered in daily life.

Allyl Compounds↗
PubMed2004

Characteristics of pancreas transplantation currently performed in japan.

The Pancreas Transplantation (tx) Program under the Japanese Organ Transplant Act was started in 2000. PTx is indicated for type 1 diabetic patients on hemodialysis therapy. As of April 2003 93 patients are listed as candidates. Ten cases of PTx with enteric or bladder-drained technique were performed during the last 3 years as well as nine SPK and one PAK. Of 10 cases, nine recipients are insulin-free with HbA1c values ranging from 4.4% to 5.7%, although exogenous insulin was required in six cases temporarily, namely for a median 63 days (12 to 225 days). One case was lost due to pancreatic graft thrombosis. All 10 kidney grafts are functioning. Based on the experiences with 14 cases of pancreas tx using non-heart-beating (NHB) donors, we defined the criteria of NHB donor as: age younger than 40 years and cessation of respiratory support. One case of SPK with graft of NHB donor was done, and the recipient is off insulin. Pancreas and kidney are allocated for SPK if the recipient shares at least one HLA-DR antigen. Marginal donors were defined as higher mean donor age, median 37 (range 18 to 58 years); mean 38 +/- 12 years), and no death cause of by trauma. The revascularization of gastroduodenal artery to the pancreatic graft was performed in eight cases to minimize the risk of ischemic injury to the pancreatic graft and technical failure in cases of marginal donor.

Adult↗
PubMed2004

RNA editing of interleukin-12 receptor beta2, 2451 C-to-U (Ala 604 Val) conversion, associated with atopy.

BACKGROUND: The production of IgE in B lymphocytes is down-regulated by IFN-gamma. IL-12 induces IFN-gamma production by T lymphocytes and natural killer cells by binding to its specific receptor. RNA editing is a post-transcriptional modification. OBJECTIVE: Here we show that the RNA editing of IL-12 receptor (R) beta2 is associated with atopy. METHODS: Atopic patients and non-atopic healthy controls were studied. Fragments of IL-12R beta2 cDNA and genomic DNA were amplified and sequenced. Furthermore, the function of the IL-12R beta2 chain was investigated. RESULTS: Sequence analysis of the cDNA clones representing IL-12R beta2 mRNA transcripts revealed a C-to-U conversion at nucleotide 2451 (Ala 604 Val) on exon 13 in some atopic patients. Surprisingly, sequence analysis of their genomic DNA showed no 2451 C-to-T (Ala 604 Val) mutation. We concluded that the observed C-to-U mismatch in the cDNA clone is due to a post-transcriptional modification, RNA editing. The C-to-U conversion was observed in 21 (20.6%) of 102 atopic patients, whereas this conversion was observed in only 4 (3.8%) of 104 non-atopic subjects (P<0.001). IFN-gamma production by peripheral blood mononuclear cells (PBMCs) stimulated with IL-12 in the subjects with the C-to-U conversion was significantly lower than that in the subjects without the C-to-U conversion. In atopic patients with the C-to-U conversion, PBMCs faintly showed the tyrosine phosphorylation of Stat4, and the IgE production by PBMCs was not suppressed by IL-12 whereas it was suppressed by IFN-gamma. CONCLUSIONS: The RNA editing of IL-12R beta2, 2451 C-to-U (Ala 604 Val) conversion causes impairment of the IL-12 signal cascade and the subsequent reduction in IFN-gamma production, resulting in the impaired down-regulation of IgE production. This is the first report indicating that atopy is associated with RNA editing.

Case-Control Studies↗
PubMed2004

Molecular analysis of B-cell differentiation in selective or partial IgA deficiency.

Selective IgA deficiency is the most common form of primary immunodeficiency, the molecular basis of which is unknown. To investigate the cause of selective IgA deficiency, we examined what stage of B-cell differentiation was blocked. DNA and RNA were extracted from three Japanese patients with selective IgA deficiency and three with a partial IgA deficiency. In selective IgA deficiency patients, Ialpha germline transcript expression levels decreased and alpha circle transcripts were not detected. Stimulation with PMA and TGF-beta1 up-regulated Ialpha germline and alpha circle transcripts. In some patients, IgA secretion was induced by stimulation with anti-CD40, IL-4 and IL-10. In partial IgA deficiency patients, Ialpha germline, alpha circle transcripts and Calpha mature transcripts were detected in the absence of stimulation. Our findings suggest that the decreased expression level of Ialpha germline transcripts before a class switch might be critical for the pathogenesis of some patients with selective IgA deficiency. However, in patients with a partial IgA deficiency, B-cell differentiation might be disturbed after a class switch.

Adolescent↗
PubMed2004

Identification of somatic and germline mosaicism for a keratin 5 mutation in epidermolysis bullosa simplex in a family of which the proband was previously regarded as a sporadic case.

Epidermolysis bullosa simplex (EBS) is an autosomal-dominant inherited blistering skin disease characterized by intraepidermal blistering due to mechanical stress-induced degeneration of basal keratinocytes. EBS is caused by mutations in either keratin 5 or keratin 14, the major keratins expressed in the basal layer of the epidermis. We experienced a unique EBS-affected family. The proband had a heterozygous 1649delG mutation in the keratin 5 gene and had been reported as a case of de novo mutation, because the mutations were not detected in the parents' DNA from blood samples. However, the proband's younger sister was revealed to have the same disease at birth and we found the same mutation in her. We reinvestigated the familial segregation of the 1649delG mutation and it was shown that the mother's DNA from hair bulb and buccal cell samples had the 1649delG mutation heterozygously, but her DNA from blood samples did not. A careful check on the mother's history disclosed that she had migratory circinate pigmentation in her skin in childhood, which means maternal somatic and germline mosaicism. The demonstration of somatic and gonadal mosaicism in the keratin 5 gene is important for accurate genetic counselling of families with sporadic cases of EBS.

Base Sequence↗
PubMed2003

Construction of a comparative RFLP map of Echinochloa crus-galli toward QTL analysis of flooding tolerance.

To analyze quantitative trait loci (QTLs) affecting flooding tolerance and other physiological and morphological traits in Echinochloa crus-galli, a restriction fragment length polymorphism (RFLP) map was constructed using 55 plants of the F(2) population ( E. crus-galli var. praticola x E. crus-galli var. formosensis). One hundred forty-one loci formed 41 linkage groups. The total map size was 1,468 cM and the average size of linkage groups was 35.8 cM. The average distance between markers was 14.7 cM and the range was 0-37.2 cM. Early comparisons to the genetic maps of other taxa suggest appreciable synteny with buffelgrass ( Pennisetum spp.) and sorghum ( Sorghum spp.). One hundred ninty-one F(2) plants were used to analyze QTLs of flooding tolerance, plant morphology, heading date, number of leaves, and plant height. For flooding tolerance, two QTLs were detected and one was mapped on linkage group 24. Other traits, including plant morphology, heading date, number of leaves, and plant height were highly correlated. Three genomic regions accounted for most of the mapped QTLs, each explaining 2-4 of the significant marker-trait associations. The high observed correlation between the traits appears to result from QTLs with a large contribution to the phenotypic variance at the same or nearby locations.

Chromosome Mapping↗
PubMed2003

The mitochondrial acetoacetyl-CoA thiolase (T2) deficiency in Japanese patients: urinary organic acid and blood acylcarnitine profiles under stable conditions have subtle abnormalities in T2-deficient patients with some residual T2 activity.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism affecting isoleucine and ketone bodies in the catabolic process. Mutation analysis and expression analysis of mutant cDNAs have facilitated the division of T2-deficient patients into two groups: those with null mutations in either allele (group 1) and those with mutation(s) retaining some residual T2 activity in at least one of two mutant alleles (group II). Among 5 Japanese T2-deficient patients, GK01 belonged to group I and the other patients (GK19, GK19B, GK30 and GK31) to group II. As we have suggested previously, the severity of ketoacidotic episodes in the group II patients was similar to that in the group I patient. However, the urinary organic acid and blood spot acylcarnitine profiles under stable conditions differed between the two groups. The group I patient had typical profiles for the T2 deficiency. In contrast, in all four patients in group II, tiglylglycine was not or was only faintly detected and the 2-methyl-3-hydroxybutyrate levels were less than the cutoff value. Their tiglylcarnitine levels were within the normal range and 2-methyl-3-hydroxy-, butyrylcarnitine was detected just around the cutoff value in our newborn screening pilot test. Hence, these analyses under stable conditions are not reliable for diagnosing the T2 deficiency in the group II patients. The T2 deficiency (group II) can be misdiagnosed as normal if these analyses are performed under nonepisodic conditions and possibly during the newborn screening for inborn errors of metabolism.

Acetoacetates↗
PubMed2003

Semiquantitative evaluation of mRNAs for the membranous form of immunoglobulin heavy chain is useful for investigating the etiology in CVID.

Common variable immunodeficiency (CVID) is a primary antibody deficiency syndrome characterized by defective B-cell maturation and antibody formation resulting in low serum antibody levels of all immunoglobulin (Ig) isotypes. To investigate the pathogenesis of CVID, we developed a set of competitive polymerase chain reaction for membrane-bound Ig heavy chain (mHC) mRNAs for IgM, IgG and IgA. Data on three children with CVID in group A of Bryant's classification were analysed. All the three mHC mRNA levels in Patient 1 were almost same as those in healthy controls. In Patient 2, mHC mRNA for IgM was detected at a level similar to that in controls, but mHC mRNAs for IgG and IgA heavy chains were not detected. In Patient 3, all the three mHC mRNAs were undetectable. Our data suggest that a different molecular basis exists in these patients with CVID even though all belong to group A of Bryant's classification. Use of our method facilitates a better understanding of molecular events in CVID patients and may be useful for precise classifications of CVID.

Child↗
PubMed2002

Molecular study of electron transfer flavoprotein alpha-subunit deficiency in two Japanese children with different phenotypes of glutaric acidemia type II.

BACKGROUND: Electron transfer flavoprotein is a mitochondrial matrix protein composed of alpha- and beta-subunits (ETF alpha and ETF beta, respectively). This protein transfers electrons between several mitochondrial dehydrogenases and the main respiratory chain via ETF dehydrogenase (ETF-DH). Defects in ETF or ETF-DH cause glutaric acidemias type II (GAII). MATERIALS AND METHODS: We investigated the molecular basis of ETF alpha deficiency in two Japanese children with different clinical phenotypes using expression study. RESULTS: Patient 1 had the severe form of GAII, a compound heterozygote of two mutations: 799G to A (alpha G267R) and nonsense 7C to T (alpha R3X). Patient 2 had the mild form and carried two heterozygous mutations: 764G to T (alpha G255V) and 478delG (frameshift). Both patients had one each of missense mutations in one allele; the others were either nonsense or truncated. Restriction enzyme digestion assay using genomic DNAs from 100 healthy Japanese revealed that these mutations were all novel. No signal for ETF alpha was detected by immunoblotting in cases of missense mutants, while wild-type cDNA resulted in expression of ETF alpha protein. Transfection with wild-type ETF alpha cDNA into cultured cells from both patients elevated incorporation of radioisotope-labelled fatty acids. CONCLUSION: These four mutations were pathogenic for GAII and missense mutations, alpha G255V and alpha G267R were considered anecdotal for mild and severe forms, respectively.

Electron-Transferring Flavoproteins↗
PubMed2001

Urinary organic acids in peroxisomal disorders: a simple screening method.

Using GC-MS, we studied urinary organic acids in 20 Japanese patients with peroxisomal disorders, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and single deficiency of peroxisomal beta-oxidation enzymes. Non-ketotic dicarboxylic aciduria with elevated sebacate/adipate molar ratio was observed in 19 of the 20 patients. Elevation of 2-hydroxysebacate and epoxydicarboxylic acids were seen in 13 and 18, respectively. Tyrosyluria was remarkable in all patients. In two ZS patients, we tracked the time course from birth to infancy, and all the above stated findings were detected, except for one sample. Urinary organic acid analysis is indeed useful for screening subjects with peroxisomal disorders.

Acids↗
PubMed2001

Inducible expression of Stat4 in dendritic cells and macrophages and its critical role in innate and adaptive immune responses.

Autocrine activation of APC by IL-12 has recently been revealed; we demonstrate here that inducible expression of Stat4 in APC is central to this process. Stat4 is induced in dendritic cells (DC) in a maturation-dependent manner and in macrophages in an activation-dependent manner. Stat4 levels directly correlate with IL-12-dependent IFN-gamma production by APC as well as IFN-gamma production by DC during Ag presentation. The Th2 cytokines IL-4 and IL-10 suppress Stat4 induction in DC and macrophages when present during maturation and activation, respectively, diminishing IFN-gamma production. In contrast, IL-4 has no effect on Stat4 levels in mature DC and actually augments IFN-gamma production by DC during Ag presentation, indicating that IL-4 acts differently in a spatiotemporal manner. The functional importance of Stat4 is evident in Stat4(-/-) DC and macrophages, which fail to produce IFN-gamma. Furthermore, Stat4(-/-) macrophages are defective in NO production in response to IL-12 and are susceptible to TOXOPLASMA: Autocrine IL-12 signaling is required for high-level IFN-gamma production by APC at critical stages in both innate and adaptive immunity, and the control of Stat4 expression is likely an important regulator of this process.

Animals↗
PubMed2001

The clinical phenotype and outcome of mitochondrial acetoacetyl-CoA thiolase deficiency (beta-ketothiolase or T2 deficiency) in 26 enzymatically proved and mutation-defined patients.

Mitochondrial acetoacetyl-CoA thiolase (T2 enzyme) deficiency (MIM 203750) is an autosomal recessive disorder of isoleucine and ketone-body metabolism. We determined the molecular basis of T2 enzyme deficiency in 26 patients at the levels of skin fibroblast enzyme activity, protein integrity, and DNA nucleotide sequence. Thirty different disease-associated alleles were identified. From these data we predicted that T2 in 6 of the 26 patients would have a mild effect on the enzyme protein and 20 would have a severe effect from their mutant genotypes. The corresponding clinical data were collected (by interviews and questionnaires) for the patients in the two groups. We found that genotype does not predict clinical severity and mutant sibs can have different clinical phenotypes; there were no consistent differences in clinical severity between patients with null-conferring or residual-conferring genotypes for T2 activity; only the absence of or a low urinary excretion of tiglyglycine during ketoacidosis correlated with a mild genotype. In general, T2 deficiency has a favorable outcome and 23 of 26 patients developed normally; one died during the first ketoacidotic episode and two have developmental delay. The median age at onset for the first ketoacidotic episode is 15 months (range 3 days to 48 months). The frequency of attacks falls with age, the last in our series occurring at 10 years of age; 11 patients had only one episode and 3 patients had none. We conclude that clinical consequences of T2 deficiency are avoidable with early diagnosis, appropriate management of ketoacidosis, and modest protein restriction.

Acetyl-CoA C-Acyltransferase↗
PubMed2001

A novel single-base substitution (380C>T) that activates a 5-base downstream cryptic splice-acceptor site within exon 5 in almost all transcripts in the human mitochondrial acetoacetyl-CoA thiolase gene.

Most mutation-related aberrant splicing occurs in the conserved splice-acceptor and -donor sites and some exonic mutations also affect splicing. We identified and characterized a point mutation (380C>T) in a Spanish patient (GK25) with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency. GK25 is a homozygote of 380C>T, which activates a cryptic splice-acceptor site 5 bases downstream from 380C>T within exon 5, causing aberrant splicing in 94% of transcripts. The aberrant splicing results in a 17-amino acids deletion, including the active-site 126Cys. The 380C>T mutation also results in A127V mutation in 6% of transcripts. Transient expression analysis showed that the A127V mutation did not retain T2 activity, indicating that 380C>T was a null mutation. Although this cryptic splice site has a higher Shapiro and Senapathy's score (86) in even a normal sequence than the authentic splice-acceptor site of intron 4 (78), it is not used in normal controls. While the 380C>T mutation increases the score slightly (90), the cryptic splice site is used in almost all transcripts in GK25 fibroblasts. This is an example in which a point mutation activates a cryptic splice-acceptor site motif that is used preferentially over the upstream authentic splice site.

Acetyl-CoA C-Acyltransferase↗