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Biomedical subjects

Y Fukumaki

Publications and source records attributed to Y Fukumaki.

At least 73 records · Page 4Linked to original sources
PubMed1994

Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase.

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91phox) and 22-kDa (p22phox) subunits, and two cytosolic factors (p47phox and p67phox), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.

Amino Acid Sequence↗
PubMed1994

Rapid and practical detection of beta-globin mutations causing beta-thalassemia by fluorescence-based PCR-single-stranded conformation polymorphism analysis.

We report a useful method for daily clinical examination for the diagnosis of thalassemia. We applied a fluorescence-based image analyser to a non-radioisotopic PCR-single-stranded conformation polymorphism (SSCP) analysis to detect mutations in the beta-globin gene. PCR primers were labelled with rhodamine X and the amplified fragments from the beta-globin gene were resolved by non-denaturing polyacrylamide gel electrophoresis. After loading, the glass plate was set in the image analyser and scanned with a green laser. We detected four common mutations in exon I and two major mutations in intron 1 of the beta-globin gene isolated from patients with beta-thalassemia. Moreover, to discriminate mutations and natural polymorphisms, we used primers including one base mismatch at the polymorphic site, which can substitute the polymorphic site by a constant base in the PCR amplified fragment. This fluorescence-based system was simple to operate and results were obtained rapidly as clear image data. Therefore, once the optimal conditions of the electrophoresis are determined, this system will be suitable for daily clinical use, especially for screening of molecular defects and for the prenatal diagnosis of genetic disorders.

Base Sequence↗
PubMed1994

Significance of pH on differentiation of human erythroid cell lines.

As physiological factors and compounds, BSA and/or higher pH of the culture medium could induce the erythroid differentiation of cells. Optimum pH values of the culture medium for higher spontaneous differentiation of KU-812 and K562 cells after 7 days cultivation were 7.5 and 7.6, respectively. The synergistic effects on the differentiation were observed by exposure to hemin under the higher pH condition. In the presence of BSA, 25% of KU-812 and 40% of K562 cells became benzidine positive. Synergistic effects of BSA and the higher pH of the medium were also observed.

Cell Differentiation↗
PubMed1994

Differential induction of adult and fetal globin gene expression in the human CML cell subline KU-812F/33.

Various chemicals which are known to have positive effects on differentiation of some erythroid cell lines were tested on a human chronic myelogenous leukemia cell line, KU-812F. Succinic acid, 5-azacytidine, daunomycin, and hemin showed a positive effect. Among them, hemin and 5-azacytidine were the most effective inducers for erythroid differentiation of KU-812F cells. Dimethylsulfoxide, cytosine arabinofuranoside, and sodium n-butyrate showed no effect. In addition, subclone KU-812F/33 derived from the KU-812F cell line showed differential expression of the beta- and gamma-globin genes in the presence of either 2 microM 5-azacytidine or 40 microM hemin. Hemoglobin synthesis in differentiated KU-812F/33 cells was analyzed by isoelectric focusing gel electrophoresis, and S1 mapping analysis of beta- and gamma-globin mRNA was performed. After treatment with 5-azacytidine, the beta-globin gene expression was predominantly enhanced (18.75-fold higher level of beta-globin mRNA). After treatment with hemin, the most notable increase was in the gamma-globin gene expression (1.83-fold higher level of gamma-globin mRNA), while no increment of beta-globin was observed.

Azacitidine↗
PubMed1994

A novel mutation in the erythrocyte protein 4.2 gene of Japanese patients with hereditary spherocytosis (protein 4.2 Fukuoka).

Human erythrocyte protein 4.2 (band 4.2; pallidin) is a major membrane protein that comprises 5% of the total weight of the human erythrocyte membrane. Deficiencies of this protein have been observed in hereditary spherocytosis with anaemia, suggesting a role of protein 4.2 in erythrocyte stability and integrity. The molecular basis of this disorder remains unknown. As a first step in elucidating the pathogenesis of hereditary spherocytosis associated with protein 4.2 deficiency, we cloned and sequenced the erythrocyte protein 4.2 gene from a normal Japanese person. We prepared sets of oligonucleotide primers for polymerase chain reaction (PCR) and determined nucleotide sequences of exons and exon-intron boundaries of the protein 4.2 gene from three unrelated Japanese patients with hereditary spherocytosis due to a complete defect of protein 4.2, using PCR-related techniques. Two patients were homozygous for a missense mutation in codon 142 with the Ala (GCT)-->Thr (ACT) amino acid substitution that has been reported previously (protein 4.2NIPPON), whereas one patient was compound heterozygous for the same missense mutation in codon 142 and a guanine-adenine transition in codon 119 that changes the codon for Trp (TGG) to the termination codon (TGA) (protein 4.2Fukuoka). No additional mutation was identified in other exons of the protein 4.2 genes. Dot-blot hybridization with allele-specific oligonucleotide probes showed that homozygosity for the missense mutation in codon 142 and compound heterozygosity for the codon 142 and the codon 119 mutations were related to protein 4.2 deficiency in the families. Although two alleles of missense mutation of the codon 142 were also detected in 100 alleles of healthy Japanese, results obtained in this study indicate that the two mutations described above are closely related to the pathogenesis of hereditary spherocytosis due to protein 4.2 defect.

Adult↗
PubMed1994

Nondeletional type of hereditary persistence of fetal haemoglobin: molecular characterization of three unrelated Thai HPFH.

The beta-globin gene clusters of three unrelated Thai families with a nondeletional type of hereditary persistence of fetal haemoglobin (HPFH) were studied using polymerase chain reaction-related techniques. All appeared to have normal nucleotide sequences from the Cap site to position -400 of both the G gamma- and A gamma-globin genes. Two individuals suspected of having a beta-thalassaemia gene linked to the high HbF condition also had a normal beta-globin gene sequence, spanning from position -108 from the Cap site to the polyadenylation site. Deletion of four nucleotides, AGCA, at positions -225 to -222 of one A gamma-globin allele was detected in one subject and was confirmed by dot-blot hybridization. Restriction fragment length polymorphisms in the beta-globin gene cluster showed that the 5' haplotype (-+-++) and the presence (+) of an Xmm 1 polymorphic site at -158 of the G gamma-globin gene are associated with the high F phenotype in these families. Direct sequencing of the 5' hypersensitive-2 (5' HS-2) site of the locus control region (LCR) showed that this Xmn 1 (+) site is also linked to a specific rearrangement of TA repeats (TA)9CACATATACG(TA)10, in HS-2 segment.

Adult↗
PubMed1993

Association of RNA with human papillomavirus E7 protein of type 16 but not type 6b.

Nonfused human papillomavirus (HPV) type 16 and 6b E7 proteins were expressed in E. coli and fractionated by ion exchange and gel filtration chromatography. The E7 protein of type 6b was purified, but that of type 16 was found to be associated with RNA molecules which could not be excluded by repeated chromatography. The type 16 E7 protein in CaSki cells was also associated with RNA molecules of the same size.

Animals↗
PubMed1993

Human fetal-to-adult globin gene switching in transgenic mice: persistent expression of the G gamma-globin gene in the Japanese HPFH.

We linked a 3.3 kb fragment containing the human gamma-globin gene together with a 5.5 kb fragment containing the human beta-globin gene. This construct was introduced into the germ line of mice. Analysis of the resulting transgenic mice showed that expression of the human gamma- and beta-globin genes were regulated as that of the mouse embryonic and adult globin genes and human gamma-to-beta globin switching was reconstructed in the mice. By replacing the gamma-globin gene in the construct with modified counterparts, this transgenic mouse system enables us to analyze cis-acting elements essential for erythroid-specific and developmental stage-specific expression of the test gene under the condition in which human beta-globin gene expression is regulated. In this system expression of the G gamma-globin gene bearing the point mutation found in a Japanese patient of hereditary persistence of fetal hemoglobin (HPFH) (1) persisted at a equivalent level to beta-globin expression in fetal and adult mice.

Aging↗
PubMed1993

Establishment and characterization of an erythropoietin-dependent subline, UT-7/Epo, derived from human leukemia cell line, UT-7.

UT-7 is a human leukemic cell line capable of growing in interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo) (Komatsu et al, Cancer Res 51:341, 1991). To study the effect of Epo on proliferation and differentiation of UT-7, we maintained the UT-7 cell culture for more than 6 months in the presence of Epo. As a result, a subline, UT-7/Epo, was established. The growth of UT-7/Epo could be supported by Epo but not by GM-CSF or IL-3. UT-7/Epo showed a greater level of heme content and ratio of benzidine-positive staining cells than did UT-7. Butyric acid promoted the synthesis of hemoglobin in UT-7/Epo, but not UT-7. Further, the mRNA concentrations of the c-myb oncogene and GM-CSF receptor beta-subunit were decreased substantially in UT-7/Epo cells. These findings showed that UT-7/Epo cells had progressed further in erythroid development than UT-7 cells, and suggested that long-term culture in Epo had promoted this differentiation. Whereas availability of the Epo receptor (Epo-R) for binding of Epo was reduced in UT-7/Epo cells compared with UT-7 cells, the Epo-R showed a similar affinity for Epo. This observation suggested that change(s) in postreceptor signaling step might be involved in the establishment and maintenance of the UT-7/Epo phenotype.

Butyrates↗
PubMed1993

PI*S(iiyama), a deficiency gene of alpha 1-antitrypsin: evidence for the occurrence in western Japan.

An alpha 1-antitrypsin deficiency associated with pulmonary emphysema was investigated in a 32-year-old Japanese male. Polymerase chain reaction (PCR)-amplified fragments and dot blot hybridization with allele-specific oligonucleotide probes revealed that the patient was homozygous for a C to T transition at codon 53, resulting in the substitution of Phe53 for Ser53 (PI*S(iiyama)). Crossed immunoelectrophoresis after isoelectric focusing and agarose gel electrophoresis showed atypical banding patterns. PI*S(iiyama) is a rare deficiency gene, but it can occur sporadically all over the Japan.

Adult↗
PubMed1993

A missense mutation in the proteolipid protein gene responsible for Pelizaeus-Merzbacher disease in a Japanese family.

We investigated the proteolipid protein (PLP) gene of two boys in a Japanese family with Pelizaeus-Merzbacher disease (PMD), an X-linked neurologic disorder characterized by dysmyelination in the central nervous system (CNS). The patients showed similar clinical signs from birth and autopsy on the elder brother confirmed a connatal type of PMD. Direct sequencing of the PLP gene and PLP mRNAs from the brain of the PMD patient revealed a G to T transition in exon V of the PLP gene, which leads to a glycine to cysteine substitution at residue 220. Allele-specific oligonucleotide hybridization revealed that this mutation was also present in his brother, but was absent in 100 X chromosomes of normal Japanese individuals. Northern blot analysis showed that the mRNA levels of PLP and myelin basic protein, two major myelin proteins produced by oligodendrocytes, were much reduced in the PMD brain, hence, there was a specific loss of oligodendrocytes. It seems likely that the substitution is responsible for PMD (connatal type) in this particular family and causes oligodendrocytes death in the CNS.

Adolescent↗
PubMed1993

A novel insertional mutation at exon VII of the myelin proteolipid protein gene in Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is an X-linked neurological disorder characterized by dysmyelination in the central nervous system (CNS). Recently mutations of the myelin proteolipid protein (PLP) gene which encodes both PLP and its isoform, DM-20 generated by alternative splicing, have been demonstrated in PMD patients. We analyzed the seven exons of the PLP gene of a Japanese boy affected with PMD by direct sequencing and identified an insertion event in exon VII of the PLP gene. This mutation was also present in his carrier mother, but was absent in ninety-five X chromosomes of normal Japanese. The frame-shift mutation leads to the production of truncated PLP with altered carboxyl terminal amino acid sequences, resulting in considerable change of the structure of PLP and DM-20 necessary for functional purposes. This is the first report of a mutation in exon VII of the PLP gene associated with PMD.

Adolescent↗
PubMed1993

Severity differences in beta-thalassaemia/haemoglobin E syndromes: implication of genetic factors.

Genetic factors determining the difference in severity of anaemia in beta-thalassaemia/HbE disease were studied in 90 patients who had haemoglobin levels, at steady state, ranging from 4.2 to 12.6 g/dl. Co-inheritance of alpha-thalassaemia 2 and haemoglobin Constant Spring could significantly decrease the severity of the disease. Inheritance of a beta-thalassaemia chromosome with Xmn I cleavage site at position -158 of the G gamma-globin gene which was linked to the haplotype -+-++ or ++-++, was associated with a milder anaemia. Two copies of these alleles were necessary to produce a significant clinical effect. Increased expression of the G gamma-globin gene and higher production of haemoglobin F, which could reduce the overall globin chain imbalance, were also associated with homozygosity for the Xmn I cleavage site and thus with less severe anaemia. However, this effect was not seen in Xmn I site heterozygotes. Whether the effects of the Xmn I polymorphism, HbF concentration and G gamma/A gamma ratio act separately or through common mechanisms in reducing anaemia remains to be ascertained.

Adolescent↗
PubMed1992

Delta-thalassemia caused by disruption of the site for an erythroid-specific transcription factor, GATA-1, in the delta-globin gene promoter.

Delta-thalassemia is a complex group of inherited disorders of globin genes characterized by impaired synthesis of the delta-globin chain. The T-C substitution was detected at position -77 of the delta-globin gene isolated from three independent Japanese individuals who were homozygotes for delta-thalassemia. To elucidate the significance of the mutation in delta-globin gene expression, we investigated the genotype of three delta-thalassemia homozygotes and 58 normal individuals using dot blot hybridization of the polymerase chain reaction (PCR)-amplified DNA. The mutation was observed in six alleles of three homozygotes, while no mutation was detected in 116 alleles of normal individuals, thereby indicating the close association of this mutation with the thalassemia phenotype. Since the mutation (TTATCT-TCATCT) is located within the inverted binding motif of GATA-1 (T or A-G-A-T-A-G or A), an erythroid cell-specific transcription factor, we did gel retardation assays using nuclear extracts from the erythroid cells. We found that GATA-1 binds the oligonucleotide spanning positions -61 to -90, but does not bind to the oligonucleotide with the mutation at position -77. Competition gel retardation assays showed that GATA-1 binding can be competed out by the fragment with the GATA-1 motif, but not with the mutant oligonucleotide. Analysis of the transient expression of the CAT gene linked to the delta-globin gene promoter region demonstrated that the construct with the mutant promoter region was expressed about 20-fold less compared with the normal one. Thus, the mutation at position -77 impairs delta-globin gene expression by abolishing GATA-1 binding to the AGATAA sequence of the promoter region of the delta-globin gene. This provides a good example of involvement of tissue-specific transacting factors in the molecular pathogenesis of hereditary diseases.

Base Sequence↗
PubMed1992

Identification of five rare mutations including a novel frameshift mutation causing beta zero-thalassemia in Thai patients with beta zero-thalassemia/hemoglobin E disease.

6 out of 14 uncharacterized beta-thalassemia alleles from 187 Thai beta-thalassemia/HbE patients were identified by direct sequencing of DNA amplified by polymerase chain reaction. A novel mutation occurring from an insertion of adenosine in codon 95, which results in a shift of the reading frame with terminator at the new codon 101, was detected in one patient. In addition, two frameshift mutations not previously reported among the Thai population were also detected in 3 patients: one with a deletion of thymidine in codon 15 and two with an insertion of cytidine in codons 27/28. A frameshift mutation that occurred from a cytidine deletion in codon 41 was also found in one patient in this study. The remaining case was an amber mutation, GAG-TAG, in codon 43 in exon 2 of the beta-globin gene. These mutations bring the number of mutations known to be present in the Thai population to a total of 20, 15 of which were detected in beta-thalassemia/HbE patients.

Adult↗
PubMed1992

Molecular basis of beta thalassemia in the south of Thailand.

A total of 103 beta thalassemia genes from 78 children (45 with Hb E/beta thalassemia, 8 with beta thalassemia heterozygotes, and 25 with homozygous beta thalassemia) were analyzed using dot-blot hybridization of the polymerase chain reaction-amplified DNA and direct DNA sequencing. Nine mutations were characterized in 98/103 (95%) of beta thalassemia alleles, of which six (a 4 bp deletion in codons 41-42, a G-C transition at position 5 of IVS-1, A-G transition at codon 19, an A-T transition at codon 17, an A-G transition at position -28 upstream of the beta globin gene, a G-T transition at position 1 of IVS-1), accounted for 92%. The spectrum of beta thalassemia mutations in Chinese Thai is similar to that reported among the Chinese from other parts of the world. The distribution of beta thalassemia mutations in Muslim Thai is similar to that reported among Malaysians. The most common beta thalassemia mutation in Thai and Chinese Thai patients is the frameshift mutation at codons 41-42, in comparison with the Muslim Thai in whom the G-C transition at position 5 of the IVS-1 mutation predominates. The heterogeneity of molecular defects causing beta thalassemia should aid in the planning of a prenatal diagnosis program for beta thalassemia in the South of Thailand.

Alleles↗