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Biomedical subjects

H Hirai

Publications and source records attributed to H Hirai.

At least 163 records · Page 9Linked to original sources
PubMed1999

Cytological, genetic and evolutionary functions of chiasmata based on chiasma graph analysis.

The nature of the chiasma as a cytological parameter for analysing cross-over was reexamined quantitatively by an improved chiasma graph method. It was reconfirmed in Mus platythrix (n =13) that interstitial chiasmata at diakinesis are distributed randomly and almost uniformly along bivalents except for the centromere and telomere regions. The size of these chiasma blank regions was consistently 0.8% of the total length of haploid autosomes in all chromosomes. There was a minimum value of chiasma interference distance between two adjacent chiasmata, which was constantly 1.8% in all chromosomes. The chiasma frequency at diakinesis was 20.1+/-2. 0 by the conventional method including terminal chiasmata. However, the primed in situ labeling technique revealed that terminal chiasmata were mostly telomere-telomere associations. From these data and also from recent molecular data we concluded that the terminal chiasma is cytologically functional for ensuring the normal disjunction of bivalents at anaphase I, but genetically non-functional for shuffling genes. The chiasma frequency excluding terminal chiasmata was 14.6+/-1.8. Reexamination of the chiasma frequency of 106 animal species revealed that the chiasma frequency increased linearly in proportion to the haploid chromosome number in spite of remarkable difference in their genome size. The increase in chiasma frequency would be evolution-adaptive, because gene shuffling is expected to be accelerated in species with high chromosome numbers.

Animals↗
PubMed1999

Transcriptional inhibition of p53 by the MLL/MEN chimeric protein found in myeloid leukemia.

The t(11;19)(q23;p13.1) translocation is frequently found in adult myeloid leukemia. In the MLL/MEN fusion protein generated by this translocation, most of the coding region of the MEN protein, an RNA polymerase II elongation factor, is fused to the N-terminal third of the MLL protein, a possible transcriptional regulator. However, the molecular mechanism of leukemogenesis by the fusion protein remains unclear. We investigated the effects of the fusion protein on p53 function using luciferase assays. Overexpression of the fusion protein suppressed the transactivation ability of p53. This negative effect of the fusion protein on p53 function was dependent on the region derived from MEN. Moreover, p53 coimmunoprecipitated with MLL/MEN as well as MEN, suggesting that the fusion protein binds to p53 through the MEN region. We found that MEN binding to p53 was mediated by its N-terminal region and repression of p53 transcriptional activity was mediated by its C-terminal region. We also found that these two functional regions were essential for the transformation of Rat1 cells mediated by MEN. Although we could not demonstrate a functional difference between MLL/MEN and MEN in this study, these data suggest that the MLL/MEN chimeric transcriptional regulator may exert its oncogenic activity by inhibiting the function of the p53 tumor-suppressor protein by binding to it. Our findings provide a novel insight into the leukemogenic mechanism exerted by the t(11;19)(q23;p13.1) translocation.

Adult↗
PubMed1999

Expression of E2A-HLF chimeric protein induced T-cell apoptosis, B-cell maturation arrest, and development of acute lymphoblastic leukemia.

The E2A-HLF fusion gene, generated by t(17;19)(q22;p13) in acute lymphoblastic leukemia (ALL), encodes a chimeric transcription factor in which the trans-activating domains of E2A are fused to the DNA-binding and dimerization domains of hepatic leukemic factor (HLF). To investigate its biological role, we generated transgenic mice expressing E2A-HLF using Ig enhancer and promoter, which direct transgene expression in cells committed to the lymphoid lineage. The transgenic mice exhibited abnormal development in the thymus and spleen and were susceptible to infection. The thymus contained small numbers of thymocytes, and TUNEL staining showed that higher population of thymocytes were undergoing apoptosis. The spleen exhibited a marked reduction in splenic lymphocytes and the flow cytometric analyses and the in vitro colony formation assays showed that the B-cell maturation was blocked at a very early developmental stage. These findings indicated that the expression of E2A-HLF induced T-cell apoptosis and B-cell maturation arrest in vivo and that the susceptibility of the transgenic mice to infection was due to immunodeficiency. Moreover, several transgenic mice developed acute leukemia, classified as T-ALL based on the surface marker analysis and DNA rearrangements, suggesting that an additional event is required for malignant transformation of lymphoid cells expressing E2A-HLF. Our findings provide insight into the biological function of E2A-HLF in lymphoid development and also its role in leukemogenesis.

Adenovirus E2 Proteins↗
PubMed1999

TT virus in bone marrow transplant recipients.

TT virus (TTV) is a newly discovered transfusion-transmissible DNA virus, which may cause posttransfusion hepatitis. The virus was detected in 12% of Japanese blood donors. The aim of the study is to investigate the prevalence and clinical influence of TTV in bone marrow transplant (BMT) recipients. Sera from 25 BMT recipients obtained 6 to 12 weeks after the transplant were examined for TTV-DNA by the seminested polymerase chain reaction. Serial samples were additionally analyzed in patients with TTV-DNA. Fifteen of 25 recipients (60%) were positive for TTV-DNA after transplant, whereas it was detected in only two of 20 BMT donors (10%). In patients positive for TTV-DNA before BMT, the amount of TTV-DNA decreased to an undetectable level during the myelosuppressed period after BMT. We also found that there was a novel group of TTV, G3, classified by the nucleotide sequences. The median peak alanine aminotransferase (ALT) levels were 135.0 IU/L and 116.5 IU/L (normal range, 4 to 36 IU/L) in TTV-positive and TTV-negative recipients, respectively. In one of the seven TTV-positive patients who developed hepatic injury (ALT > 150 IU/L), a serial change in the serum TTV titer showed a good correlation with the ALT level. We concluded that (1) the prevalence of TTV is high in BMT recipients, (2) TTV might be replicated mainly in hematopoietic cells, (3) transfusion-transmitted TTV may cause persistent infection, (4) a novel genetic group of TTV, G3, was discovered, and (5) TTV does not seem to frequently cause hepatic injury, although one patient was strongly suggested to have TTV-induced hepatitis.

Adolescent↗
PubMed1999

Trans-activator Tax of human T-cell leukemia virus type 1 enhances mutation frequency of the cellular genome.

Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a potent trans-activator of specific sets of target genes, and on the other hand, it is a trans-repressor of other sets of genes. It is also an inhibitor of the tumor suppressor protein p16(INK4a) and thus has been thought to contribute to induction of adult T-cell leukemia (ATL). We examined the mutagenic effects of Tax on a cellular gene, hypoxanthine guanine phosphoribosyltransferase (hprt), and LacI gene in lambda shuttle vector exogenously integrated in Big Blue Rat-2 (BBRat-2) cells. Expression of Tax in BBRat-2 cells enhanced the frequency of HPRT(-) phenotype severalfold. Tax-expressing cell clones, BBTax-1 and -2 established from BBRat-2 cells, gave rise to an average mutation frequency of 5.9 x 10(-5) in LacI gene, but Tax-negative cell clones, BBRat-C1 and -C2, showed 2. 1 x 10(-5). The 2.8-fold increase in mutation frequency in the presence of Tax indicates that Tax expression enhanced mutation frequency in chromosomal DNA. However, neither the mutation spectrum of base transitions, transversions, and deletions/insertions nor the loci of the mutations were significantly affected by Tax expression. These findings indicate that Tax has the capability to induce random mutations and suggest that Tax would be able to modulate cellular phenotypes through mutation of the cellular genome.

Animals↗
PubMed1999

Regulation of phosphatidylcholine biosynthesis by mGluR1alpha expressed in human embryonic kidney 293 cells--A 31P-NMR study.

A recent report has demonstrated that inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release plays a crucial role in neurite growth. Here, using 31P-NMR, we examine whether activation of the metabotropic glutamate receptor 1 (mGluR1), which induces the production of IP3, could modulate phospholipid metabolism in human embryonic kidney 293 cells. mGluR1alpha- but not ionotropic glutamate receptor 1-expressing cells stimulated with glutamate exhibited a drastic reduction in the phosphorylcholine level, with corresponding increases in the level of phosphatidylcholine, a major phospholipid component. Quantitative analysis of cell growth revealed that mGluR1alpha-expressing cells cultured with 100microM glutamate were statistically significantly longer than the nontransfected cells. The effect was no longer observed following coincubation with a metabotropic glutamate receptor antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine. These results suggest that mGluR1alpha activation triggers phosphatidylcholine biosynthesis and may contribute to neurite extension.

Cell Line↗
PubMed1999

Prevalence of myopia and refractive changes in students from 3 to 17 years of age.

We investigated changes in the prevalence of myopia and mean changes in refractive errors in Japanese students from 3 to 17 years old from 1984 to 1996. Mass ophthalmologic surveys were performed annually during the course of the study. The age-specific frequency distribution of refractive errors remained similar for 6-year-old students (defined in this study as students in the first grade of primary school) during the 13-year period, but the distribution became gradually skewed toward myopia for 12-year-old students (defined in this study as students in the first grade of junior high school). Comparisons between 1984 and 1996 examinations showed a considerable increase in the incidence of myopia among those 7 years of age or older, and changes in mean refractive errors also demonstrated a greater shift toward myopia, especially in students older than 10 years, for whom the changes were statistically significant. In this 13-year period, the prevalence of myopia increased from 49.3% to 65.6% in 17-year-old students. In addition to the annual mass ophthalmologic examinations, we also performed a longitudinal 6-year study of 346 students who entered junior high school in 1989, 1990, or 1991. Among these students, the prevalence of myopia increased from 43.5% at 12 years of age to 66.0% at 17 years of age. These 346 students were divided into the following eight groups according to their refractive error (spherical power [D]) at 12 years of age: +1 D, 0 D, -1 D, -2 D, -3 D, -4 D, -5 D, and -6 D. Mean progressions of myopia in these students were as follows: for the +1 D group, -0.14 D/year; for the 0 D group, -0.25 D/year; for the -1 D group, -0.37 D/year; for the -2 D group, -0.40 D/year; for the -3 D group, -0.29 D/year; for the -4 D group, -0.25 D/year; for the -5 D group, -0.14 D/year; and for the -6 D group, -0.22 D/year. Boys and girls demonstrated a statistically significant difference in mean changes in refractive errors at the 6-year follow-up examination: the mean change in refractive error was -1.41 +/- 1.25 D for boys as compared with -1.03 +/- 1.07 D for girls (unpaired Student's t-test, P < 0.0001). Our results demonstrated an early age at onset for myopia and a recent increase in the proportion of myopic students. Further studies are needed to shed light on the extent to which myopia is caused by environmental factors, because it is through these factors that the prevalence rate may be affected.

Adolescent↗
PubMed1999

The clustering of NMDA receptor NR1 subunit is regulated by the interaction between the C-terminal exon cassettes and the cytoskeleton.

The clustering of neurotransmitter receptors at postsynaptic sites is considered to play an important role in modulating synaptic efficacy. To investigate the mechanisms underlying neurotransmitter receptor clustering, we expressed the NMDA (N-methyl-D-aspartate) receptor NR1A subunit in human embryonic kidney (HEK) 293 cells. As previously shown, the cells exhibited subcellular clusters of the receptor protein with a mean diameter of approximately 0.7 microm. To examine the involvement of cytoskeletal structures on this clustering, we disrupted actin filaments or microtubules by treating the cells with alkaloids. In the actin filament-disrupted cells, the receptor protein shifted from the cellular membrane to the cytoplasm where it formed macroclusters (approximate diameter 3 microm). In the microtubule-disrupted cells, the subcellular clusters of NR1A could not be detected and the protein was diffusely distributed throughout the cytoplasm. Similar results were obtained by coexpression of the receptor protein with fusion proteins harboring various C-terminal exon cassettes. These results suggest that subcellular clustering of the NR1 subunit of the NMDA receptors is regulated by the interaction of its C-terminus with cytoskeletal components, where differentially spliced cassettes interact separately with actin filaments or microtubules. Modulation of the interaction between the neurotransmitter receptors and the cytoskeleton leads to the rearrangement of the receptor clusters and may contribute to certain types of synaptic plasticity.

Alkaloids↗
PubMed1999

Interaction of the C-terminal domain of delta glutamate receptor with spectrin in the dendritic spines of cultured Purkinje cells.

The interaction of neurotransmitter receptors with the underlying cytoskeleton via subsynaptic proteins is an important mechanism for the targeting of the receptors to synapses in the central nervous system. We show that delta glutamate receptors (delta receptors), expressed predominantly in the dendritic spines of cerebellar Purkinje cells, directly interact with spectrin, a member of the actin-binding family of proteins. Moreover, the interaction between spectrin and C-terminal domain of the delta receptor is 50% inhibited by 1 microM of Ca2+ in vitro, compared with that in the absence of Ca2+. These results suggest that delta receptors on the postsynaptic membrane of the dendritic spines of cerebellar Purkinje cells are anchored to the actin cytoskeleton via spectrin, and that Ca2+ elevation in the dendritic spines causes delta receptor declustering by dissociation of the receptors from spectrin. This mechanism for receptor anchoring at postsynaptic sites may regulate synaptogenesis and/or synaptic plasticity.

Animals↗
PubMed1999

Rapid HLA class I DNA typing using microtiter plate-reverse hybridization assay (MRHA) by simple thermoregulation: high-resolution subtyping of the HLA-A2 and -B40 antigen groups.

We have established a precise, rapid, simple and economical subtyping method for alleles encoding the HLA-A2 and -B40 antigens using microtiter plate-reverse hybridization assay (MRHA), which is based on the general principle of HLA oligotyping by reverse dot blot hybridization. Amino-modified sequence-specific oligonucleotide (SSO) probes were immobilized covalently onto a carboxylate-modified microtiter plate. In order to perform high-resolution subtyping of the HLA-A2 and -B40 antigen groups, the alpha1 and alpha2 domain regions were amplified using a pair of group-specific primers composed of an unlabeled sense primer and a biotinylated antisense primer. PCR-amplified products were hybridized with SSO probes in hybridization buffer containing formamide for 1 hour at 37 degrees C. After washing with 2 X SSC at room temperature, the bound PCR products were detected by alkaline phosphatase-conjugated streptavidine followed by color development. All of 8 HLA-B40 suballeles, all of 2 HLA-B47 suballeles (B40 group-specific primers used in this study allowed also B47 amplification) and 17 out of 21 HLA-A2 suballeles were discriminated. The remaining four HLA-A2 suballeles were determined by analysis after exon 4 amplification. HLA-DNA typing by this method was easily and exactly performed regardless of sample number. The greatest advantages of this technique are strong positive signals obtained, reproducibility and the ease of thermoregulation for hybridization and washing as compared to previously reported microtiter plate hybridization methods.

DNA Probes↗
PubMed1999

Signal transduction pathways in normal human monocytes stimulated by cytokines and mediators: comparative study with normal human neutrophils or transformed cells and the putative roles in functionality and cell biology.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL) -3 induced tyrosine phosphorylation of 92-kDa protein in normal human monocytes. We identified this 92-kDa protein as STAT5, but not as STATs1, 3, and 6 nor c-fes and vav protooncogene products, and demonstrated its translocation to the nucleus, enhancement of specific DNA binding capacity, and potentiation of trancriptional activity by GM-CSF. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA) induced tyrosine phosphorylation of 42- and 44-kDa proteins, which were identified as extracellular signal-regulated kinase (ERK), in human monocytes. In marked contrast to neutrophils and MO7e cells, GM-CSF did not induce tyrosine phosphorylation and activation of ERK in monocytes. Among upstream signaling molecules of ERK, Shc was constitutively associated with Grb2 and was not tyrosine-phosphorylated by GM-CSF and FMLP, and Sos1 and c-Raf-1 were not phosphorylated by GM-CSF, IL-3, TNF, and FMLP in monocytes, whereas all these signaling molecules were affected and/or utilized by GM-CSF in MO7e cells. In contrast to neutrophils, p38 was constitutively phosphorylated and agonist-dependent phosphorylation and activation was not detected in human monocytes. Superoxide release stimulated by FMLP was inhibited partially by PD98059 or SB203580, a specific inhibitor of ERK or p38 pathway, and was almost completely inhibited by the combination of both inhibitors, whereas PMA-induced superoxide release was resistant to these two inhibitors in monocytes. PD98059 inhibited GM-CSF-dependent proliferation of MO7e cells. Present results indicate trancriptional roles of STAT5 and functional roles of ERK and/or p38 in normal human monocytes stimulated by physiological receptor-mediated agonists GM-CSF and FMLP. Possible roles of ERK in proliferation of transformed cells were also suggested.

Adult↗
PubMed1999

Intracellular signalling pathways of okadaic acid leading to mitogenesis in Rat1 fibroblast overexpressing insulin receptors: okadaic acid regulates Shc phosphorylation by mechanisms independent of insulin.

Okadaic acid is a powerful inhibitor of serine/threonine protein phosphatases 1 and 2A. Although it is known as a potent tumour promoter, the intracellular mechanism by which okadaic acid mediates its mitogenic effect remains to be clarified. We investigated the effect of okadaic acid on the activation of mitogenesis in Rat1 fibroblasts overexpressing insulin receptors. As previously reported, insulin induced Shc phosphorylation, Shc-Grb2 association, MAP kinase activation, and BrdU incorporation. Okadaic acid also stimulated tyrosine phosphorylation of Shc and its subsequent association with Grb2 in a time- and dose-dependent manner without affecting tyrosine phosphorylation of insulin receptor beta-subunit and IRS. However, to a lesser extent, okadaic acid stimulated MAP kinase activity and BrdU incorporation. Interestingly, preincubation of okadaic acid potentiated insulin stimulation of tyrosine phosphorylation of Shc (213% of control), Shc-Grb2 association (150%), MAP kinase activity (152%), and BrdU incorporation (148%). These results further confirmed the important role of Shc, but not IRS, in cell cycle progression in Rat1 fibroblasts. Furthermore, serine/ threonine phosphorylation appears to be involved in the regulation of Shc tyrosine phosphorylation leading to mitogenesis by mechanisms independent of insulin signalling.

Adaptor Proteins, Signal Transducing↗
PubMed1999

The transcription factor Evi-1.

Evi-1 is a transcription factor with two sets of zinc finger domains. The temporally and spatially restricted pattern of Evi-1 expression in embryonic tissues suggests a role of Evi-1 in organogenesis and morphogenesis in mouse development. Mice lacking Evi-1, which die within the first few weeks of life with multiple defects in embryonic development, suggest that Evi-1 is essential for developmental cell proliferation, vascularization, and cell-specific signaling at midgestation. In hematopoietic cells, Evi-1 expression is restricted at a transient stage of myeloid cell differentiation. Constitutive expression of Evi-1 in hematopoietic cells, which is caused by retroviral insertions or chromosomal translocations and inversions, is closely associated with myelogenous leukemias and myelodysplastic syndromes in mice and humans. In the aspect of potential therapeutic approaches, some pharmaceutical drugs or antisense oligonucleotides that repress Evi-1 expression would be useful for the treatment of Evi-1-induced neoplastic tumors.

Animals↗
PubMed1999

Genomic differentiation of 18S ribosomal DNA and beta-satellite DNA in the hominoid and its evolutionary aspects.

The chromosome localization of two human multisequence families, rDNA and beta-satellite (beta-sat) DNA, was determined in humans and apes using double color fluorescence in-situ hybridization. Both DNA probes showed a distinct hybridization pattern with species-specific variations in hominoids. The stepwise differentiation of the integration, amplification, multilocalization, and reduction of the DNAs were observed interspecifically through the seven species examined. The stepwise events allowed us to trace back a phylogenetic divergence of the hominoid at the cytogenetic level. The manifestation of the events revealed that variations of the Y chromosome and acrocentric autosomes were synapomorphic characters in the divergence and those of metacentric autosomes were autapomorphic characters. Multilocalization of rDNA in the hominoid could also be interpreted as a result of translocations in terms of hetero-site crossover followed by a centric fission and formation of an acrocentric chromosome. Based on the observed rearrangements of rDNA and beta-sat DNA, we propose the following chromosomal phylogenetic divergence order in hominoids: gibbon-siamang-orangutan-gorilla-human-chimpanzee-bonobo. Our data provide additional evidence that evolution of the hominoid can be effectively studied using cytogenetic approaches.

Animals↗