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Biomedical subjects

Y Fukumaki

Publications and source records attributed to Y Fukumaki.

At least 55 records · Page 3Linked to original sources
PubMed1996

Assembly and activation of the phagocyte NADPH oxidase. Specific interaction of the N-terminal Src homology 3 domain of p47phox with p22phox is required for activation of the NADPH oxidase.

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b558 comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (KD = 0.34 microM). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro156-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp193 by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.

Cytochrome b Group↗
PubMed1996

Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding genes.

Three Trimeresurus okinavensis (To; himehabu snake, Crotalinae) venom gland phospholipase A2 (PLA2) isozymeencoding genes, gPLA2-o1, gPLA2-o2 and gPLA2-o3, were isolated from its genomic DNA library. The nucleotide (nt) sequence analysis revealed that two of the three genes (gPLA2-o2 and gPLA2-o3) occasionally have been converted to inactivated genes by introduction of one base insertion or substitution. It was confirmed from Southern blot analysis that the To haploid genome contains only three venom gland PLA2 isozyme genes herein isolated. Comparison of these genes showed that nonsynonymous nt substitutions have occurred more frequently than synonymous nt substitutions in the protein-coding regions, except for the signal-peptide coding domain, implying that To venom gland PLA2 isozyme genes have evolved via accelerated evolution. Such an evolutionary feature of To venom gland PLA2 isozyme genes proves the general universality of accelerated evolution previously drawn for venom gland PLA2 isozyme genes of other crotalinae snakes. The variability in the mature protein-coding regions of three To venom gland PLA2 isozyme genes appears to have been brought about by natural selection for point mutations.

Amino Acid Sequence↗
PubMed1996

[Molecular mechanisms of globin gene regulation and disregulation].

Human globin genes are expressed in tissue and developmental stage specific manners. Various experiments including expression studies using the transgenic mouse system have been done to elucidate molecular mechanisms of globin gene regulation. Cis-elements and transcription factors, especially erythroid specific and stage specific factors, involved in proper globin gene regulation are summarized. A model of Hb switching is discussed in terms of the interaction of LCR with each globin gene region. Molecular mechanisms deteriorating or enhancing transcription of the globin genes in diseases such as thalassemia and HPFH, are also mentioned.

Animals↗
PubMed1995

Mode of activation of the GC box/Sp1-dependent promoter of the human NADH-cytochrome b5 reductase-encoding gene.

Many eukaryote promoters, particularly those for so-called housekeeping genes, have multiple GC boxes which are the binding sites of the transcription factor, Sp1. It has been proposed that Sp1 binds to the multiple GC boxes, and then the GC box-bound Sp1 interact with each other to synergistically stimulate transcription. Here, we describe a Sp1-dependent promoter which does not necessarily fit the synergistic activation mechanism. The promoter of the human NADH-cytochrome b5 reductase-encoding gene (CYTB5R) possesses five potential GC box sequences. Deletion and mutagenesis studies coupled with CAT assays revealed that three out of five GC box-like sequences were functionally active and activated transcription additively (rather than synergistically). Our results suggested that Sp1-mediated activation of transcription occurs in a promoter context-dependent manner.

Animals↗
PubMed1995

Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes.

The nucleotide sequences of four genes encoding Trimeresurus gramineus (green habu snake, crotalinae) venom gland phospholipase A2 (PLA2; phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) isozymes were compared internally and externally with those of six genes encoding Trimeresurus flavoviridis (habu snake, crotalinae) venom gland PLA2 isozymes. The numbers of nucleotide substitutions per site (KN) for the noncoding regions including introns were one-third to one-eighth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions of exons, indicating that the noncoding regions are much more conserved than the protein-coding regions. The KN values for the introns were found to be nearly equivalent to those of introns of T. gramineus and T. flavoviridis TATA box-binding protein genes, which are assumed to be a general (nonvenomous) gene. Thus, it is evident that the introns of venom gland PLA2 isozyme genes have evolved at a similar rate to those of nonvenomous genes. The numbers of nucleotide substitutions per nonsynonymous site (KA) were close to or larger than the KS values for the protein-coding regions in venom gland PLA2 isozyme genes. All of the data combined reveal that Darwinian-type accelerated evolution has universally occurred only in the protein-coding regions of crotalinae snake venom PLA2 isozyme genes.

Amino Acid Sequence↗
PubMed1995

Alpha B-crystallin in C6 glioma cells supports their survival in elevated extracellular K+: the implication of a protective role of alpha B-crystallin accumulation in reactive glia.

It has been shown by immunohistochemical studies that alpha B-crystallin accumulates in the reactive and neoplastic glial cells in a variety of pathologic situations. However, the molecular mechanism for the induction of alpha B-crystallin in diseased brains is still unknown. Since any destructive brain lesions cause an abnormal elevation in the potassium (K+) concentration of the extracellular space, which disturbs the regulatory mechanism of glial cell volume, we investigated the influence of elevated extracellular K+ on the expression of alpha B-crystallin in glial cells. The treatment of rat C6 glioma cells with augmented K+ in the culture media induced an accumulation of alpha B-crystallin mRNA in a dose-dependent manner and an accumulation of the alpha B-crystallin as well. Furthermore, an overexpression of alpha B-crystallin in the C6 transformant transfected with a rat alpha B-crystallin cDNA conferred a resistant phenotype against the insult of elevated extracellular K+ on the glioma cells. Thus, alpha B-crystallin may contribute to the survival of reactive glia in the presence of a high extracellular K+ concentration.

Animals↗
PubMed1995

Structures of genes encoding TATA box-binding proteins from Trimeresurus gramineus and T. flavoviridis snakes.

A cDNA encoding the Trimeresurus gramineus (Tg; green habu snake) TATA-box-binding protein (TgTBP) was cloned and sequenced. The cDNA encodes a 33-kDa protein with an extensive sequence similarity to those derived from other organisms, except for the N-terminal domain. Genes encoding TgTBP and Trimeresurus flavoviridis (Tf; habu snake) TBP (TfTBP) were isolated using a TgTBP cDNA and their nt sequences were determined. They are the first TBP genes entirely sequenced in higher animals. Both genes span over 15 kb and are constructed from eight exons and seven introns. Comparison of the loci of introns on the aligned amino-acid sequences of TBP from six organisms (Tg, Tf, mouse, Arabidopsis thaliana, Schizosaccharomyces pombe and Acanthamoeba castellanii) indicated that there are three highly conserved loci in the C-terminal domain.

Amino Acid Sequence↗
PubMed1995

A novel mutation causing an aberrant splicing in the protein 4.2 gene associated with hereditary spherocytosis (protein 4.2Notame).

We investigated a Japanese patient with protein 4.2 deficiency. SDS-PAGE showed a complete deficiency of protein 4.2, while Western blot analysis revealed a marked decrease in the amount of protein 4.2, and the existence of a doublet of 74 and 72 kDa bands. Direct sequencing and dot-blot hybridization with allele-specific oligonucleotide probes indicated that the proband was compound heterozygous for a missense mutation in codon 142 with Ala-->Thr (GCT-->ACT) and a single nucleotide substitution (G-->A) of the first base of intron 6 (G-->A) of the protein 4.2 gene. The former is the commonest mutation observed in cases of protein 4.2 deficiency, whereas the latter is a novel mutation, located within the consensus sequence of the 5' splicing site (AGGU) (Protein 4.2Notame). RT-PCR analysis using total RNA isolated from reticulocytes of the proband revealed that the intron 6 donor site mutation causes an abnormal splicing; exon 6 is spliced out with intron 6. The abnormal mRNA has a premature termination codon, as the result of a frameshift, and this instability may lead to degradation. Thus, there is a close relation between this mutation and the molecular pathogenesis of protein 4.2 deficiency.

Base Sequence↗
PubMed1995

A role for the distal CCAAT box of the gamma-globin gene in Hb switching.

Hereditary persistence of fetal hemoglobin (HPFH) is a condition manifested by continued expression of the gamma-globin gene in adult life. The four types of HPFH mutations (-117 G-A, -114 C-T, -114 C-G, and 13 bp del) clustered in the vicinity of the distal CCAAT box of the gamma-globin gene suggest the relevance of this region to the fetal-to-adult hemoglobin switching. Functional analysis in erythroid cell lines showed that these HPFH mutations reduced the activity of the gamma-globin promoter in K562 expressing embryonic and fetal globin chains, but not in KU812 expressing fetal and adult globin chains. These results suggest that the distal CCAAT box region has different functions in the fetus and the adult. In vitro, binding of NFE3 to the distal CCAAT box was commonly reduced by these HPFH mutations, suggesting that the binding of NFE3 to the distal CCAAT box may repress the gamma-globin gene in adults. Overlap of the binding sites of NFE3 and CP1, a potential activator, indicates the regulation of the gamma-globin gene by the competition between these factors. In addition, an unknown factor interacting with the -114 C-T HPFH mutant CCAAT box may be involved in elevation of the gamma-globin expression in an individual with the -114 C-T mutation.

Base Sequence↗
PubMed1995

Detection of alpha-thalassemia-1 (Southeast Asian type) and its application for prenatal diagnosis.

A simple non-radioactive method based on the polymerase chain reaction was used to detect the Southeast Asian type of alpha-thalassemia 1 (--). Three oligonucleotide primers, one of which was adjacent to the breakpoint of the alpha-thalassemia-1 allele, were used to amplify the 570 and 194 bp DNA fragments. The 570 bp product was specific to the alpha-thalassemia-1 determinant and the 194 bp fragment was amplified from either the alpha-thalassemia-2 (-alpha) or normal alpha-globin (alpha alpha) determinants. In Hb Bart's hydrops fetalis (--/--), only the 570 bp fragment was obtained, whereas the 194 bp fragment was amplified in normal individual (alpha alpha/alpha alpha) and alpha-thalassemia-2 trait (-alpha/alpha alpha). Both 570 and 194 bp fragments were detected in alpha-thalassemia-1 trait (--/alpha alpha) and Hb H patients (--/-alpha). This procedure is useful for the rapid screening of alpha-thalassemia-1 trait and prenatal diagnosis of Hb Bart's hydrops fetalis in populations with a high frequency of the Southeast Asian Type of alpha-thalassemia-1.

Asia, Southeastern↗
PubMed1995

Detection of varicella-zoster virus genome having a PstI site in the ocular sample from a patient with acute retinal necrosis.

We detected the virus genome in ocular samples from a 65-year-old woman with clinically diagnosed acute retinal necrosis using DNA amplification. She exhibited occlusive retinal vasculitis, confluent necrotizing retinitis, mainly peripheral, and iridocyclitis. For DNA amplification, we used recently published primers specific for varicella-zoster virus (VZV) and herpes simplex virus. Using VZV primers, we detected the VZV genome in the aqueous humor, but not in the vitreous, by amplifying a DNA fragment 642 base pairs in length. HSV DNA was not detected. After detecting the VZV genome, PstI restriction endonuclease was used because an epidemiological study found that about 25% of the VZV strains in Japan carry a mutation lacking a PstI recognition site. The VZV genome from the patient had a PstI cleavage pattern, while the positive control had a VZV genome that carried a PstI-site-less mutation. We considered our patient with acute retinal necrosis to be infected with VZV having a PstI site.

Aged↗
PubMed1995

Molecular analysis of the human fetal-to-adult globin switching.

Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by continued expression of the gamma-globin gene in adult life. Analysis of a Japanese HPFH family had revealed that the C-T transition at position -114 within the distal CCAAT box of the gamma-globin gene associated with the HPFH allele. In the vicinity of the distal CCAAT box, other two mutations (-117 C-T, 13 bp del) had been identified in individuals with a HPFH phenotype. Functional analysis of these mutant promoters in erythroid cell lines suggested that the distal CCAAT box works positively in the fetus but negatively in the adult on the expression of the gamma-globin gene. Further study on transgenic mice showed that the -114 mutation was responsible for the elevated expression of the gamma-globin gene in the adult. To elucidate the molecular mechanism underlying the persistent expression of the gamma-globin genes associated with the HPFH mutations, interaction of the mutant promoters with nuclear factors was analyzed. Relevance of the nuclear factor, NFE3, to the gamma-globin regulation was suggested by the affected binding of NFE3 to the altered distal CCAAT boxes with HPFH mutations (-117, -114, 13 bp del).

Adult↗
PubMed1995

The spectrum of beta-thalassemia mutations in southern Thailand.

Beta-thalassemia mutations in 282 alleles of 253 unrelated individuals originating from various provinces in the south of Thailand were characterized by dot blot hybridization, specific PCR-amplification and direct DNA sequencing. It was possible to characterize the mutations in 274 (97.2%) of alleles studied. Twelve different point mutations and two different large deletions of the beta-globin gene were identified. Seven common mutations, namely 4 bp deletion at codons 41/42. IVS1 position 5 (G-C), codon 19 (AAC-AGC), codon 17 (AAG-TAG), IVS1 position 1 (G-T), position -28 (A-G) and 3.5 kb deletion, accounted for about 91.5%. The mutations at mRNA cap site + 1 (A-C) and IVS1 position 1 (G-A), previously undescribed in Thailand, were found in 1 and 2 individuals, respectively. A novel mutation of 105 bp deletion at the 5' end of beta-globin gene was detected in a family originating from this area. The knowledge from this study should be useful for planning of genetic counseling and prenatal diagnosis programs for patients with beta-thalassemia in the south of Thailand.

Alleles↗
PubMed1995

Molecular mechanisms of thalassemia in southeast Asia.

Hemoglobinopathies are the most common genetic disorders in Southeast Asia. alpha-Thalassemia is most often due to a alpha-globin gene deletion. Hb Constant Spring (CS) occurs from the mutation at the termination codon of the alpha-globin gene resulting in an elongated polypeptide; alpha(CS)-globin mRNA is also unstable and only small amounts of Hb CS are produced. Thus Hb CS has an alpha-thalassemia 2-like effect. beta-Thalassemia results from a variety of molecular mechanisms, most of which are single base substitutions or deletions or insertions of one to four nucleotides. Hemoglobin E occurs from a Glu --> Lys substitution at position 26 of the beta-globin chain. The abnormal gene also results in reduced amounts of beta E-mRNA and hence of beta E-globin chains. Therefore, Hb E has a mild beta + thalassemia phenotype. Homozygous beta-thalassemia and beta-thalassemia/Hb E are the major beta-thalassemic syndromes in Southeast Asia. In spite of seemingly identical genotypes, severity of beta-thalassemia/Hb E patients can vary greatly. Some may have a severe clinical disorder approaching that seen in homozygous beta-thalassemia. A number of genetic factors have been shown to determine the differences in severity of anemia in beta-thalassemia/Hb E, including co-inheritance of alpha-thalassemia determinants and co-inheritance of other determinants which elevate Hb F expression. A correlation between the extent of beta E-globin mRNA cryptic splicing and the severity of anemia in beta(zero)-thalassemia/Hb E patients has been observed. Complete characterization of mutations causing hemoglobinopathies will help to bolster the establishment of prenatal diagnosis of these genetic disorders in the region.

Alternative Splicing↗
PubMed1995

Role of alternatively spliced beta E-globin mRNA on clinical severity of beta-thalassemia/hemoglobin E disease.

In spite of seemingly identical genotypes, severity of beta-thalassemia/hemoglobin (Hb) E patients can vary greatly. Some may have a severe clinical disorder approaching that seen in homozygous beta-thalassemia. Since mutation in codon 26 of the beta E-globin gene can lead to an alternative splicing, Hb E acts like a mild beta(+)-thalassemia. Variation in the amount of beta E-globin mRNA may also govern the difference in severity of anemia in beta-thalassemia/Hb E patients who otherwise have the same genetic determinants. We have determined the percentage of the alternatively spliced beta E-globin mRNA by the RT-PCR technique in 14 patients and found that the amount of abnormal spliced beta E-globin mRNA in those patients with severe symptoms ranged between 2.9 to 6.1%, whereas those with milder symptoms had the values which ranged between 1.6 to 2.6%. The extent of beta E-globin mRNA cryptic splicing was better associated with clinical severity of the patients than did the patterns of the Xmn I polymorphism at position -158 of the G gamma-globin gene or levels of Hb F.

Adolescent↗
PubMed1995

A simple non radioactive method for detecting beta-thalassemia/hbe disease: application to prenatal diagnosis.

We have developed allele specific polymerase chain reaction (ASPCR) that allows rapid screening of the beta E-globin and common beta-thalassemia genes in Thailand. These non-radioactive methods are based on the amplification by the polymerase chain reaction of the beta E and beta-thalassemia specific DNA fragments using specific primers. With this approach, both heterozygote and homozygote for the disease could readily be identified on agarose gel electrophoresis of the amplified DNA. We have applied the method for a prenatal diagnosis of beta-thalassemia/HbE disease in a Thai family at the second trimester of pregnancy. The result obtained was comparable to that of conventional dot blot hybridization using radioactive probes. The simplicity, accuracy and non isotopic of the approach make it a highly promising method for a carrier screening and a prenatal diagnosis of this common disorder.

Alleles↗
PubMed1994

Cloning and sequencing of the cDNA encoding human glutaredoxin.

Glutaredoxin (thioltransferase) is a small, heat-stable protein, which is involved in thiol/disulfide exchange reactions. We have isolated a cDNA that encodes glutaredoxin from a human brain cDNA library. The encoded protein contains 106 amino acids with a calculated molecular mass of 11.76 kDa and an isoelectric point of 8.09. The amino acid sequence deduced from the cDNA is more than 80% identical to those of other mammalian glutaredoxins.

Amino Acid Sequence↗