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Biomedical subjects

Y Yazaki

Publications and source records attributed to Y Yazaki.

At least 289 records · Page 16Linked to original sources
PubMed1997

Cell density-dependent apoptosis in HL-60 cells, which is mediated by an unknown soluble factor, is inhibited by transforming growth factor beta1 and overexpression of Bcl-2.

We report a novel mode of apoptosis induction observed in human leukemic HL-60 cells. These cells spontaneously underwent apoptosis in the course of proliferation when the cell density became higher than 1 x 10(6)/ml. This occurred under ordinary in vitro culture conditions, with or without fetal calf serum. Even the low density cells were committed to undergo apoptosis if they were cultured under artificially concentrated conditions. Replacement of the culture supernatant of the low density cells by that of the high density ones resulted in apoptosis induction in the former cells. This apoptosis-inducing activity of the high density cell culture supernatant was completely eliminated by the action of trypsin but was fully restored following ultrafiltration by 3-kDa pore-sized membrane. A strong apoptosis-inducing activity was recovered from the culture supernatant of the high density HL-60 cells at a specific fraction in reverse-phase column chromatography. Neither an interleukin-beta converting enzyme inhibitor nor CPP-32 inhibitor blocked the induction of cell density-dependent apoptosis in HL-60 cells, although overexpression of Bcl-2 protein markedly attenuated the induction of this mode. Surprisingly, transforming growth factor-beta1 and activin A did not induce but, rather, inhibited the induction of cell density-dependent apoptosis. These data suggest that HL-60 cells release an unknown low molecular weight peptide-containing factor in response to an increase in cell density to induce apoptosis in an autocrine manner and that the interleukin-beta converting enzyme-independent intracellular machinery for this mode of apoptosis is strongly affected by signaling events through the transforming growth factor-beta1 receptor and by the action of Bcl-2 oncoprotein.

Activins↗
PubMed1997

Role of the C terminus in histamine H2 receptor signaling, desensitization, and agonist-induced internalization.

To evaluate the role of the histamine H2 receptor C terminus in signaling, desensitization, and agonist-induced internalization, canine H2 receptors with truncated C termini were generated. Wild-type (WT) and truncated receptors were tagged at their N termini with a hemagglutinin (HA) epitope and expressed in COS7 cells. Most of the C-terminal intracellular tail could be truncated (51 of 70 residues, termed T308 mutant) without loss of functions: cAMP production, tiotidine binding, and plasma membrane targeting. In fact, the T308 mutant produced more cAMP than the WT when cell-surface expression per cell was equivalent. Pretreatment of cells with 10(-5) M histamine desensitized cAMP productions via WT and T308 receptors to similar extents. Incubation of cells expressing WT receptors with 10(-5) M histamine reduced cell-surface anti-HA antibody binding by approximately 30% (by 30 min, t1/2 approximately 15 min), but did not affect the Bmax of tiotidine in membrane fractions, which represents total receptor amounts, suggesting that WT receptors were internalized from the cell surface. In contrast, no internalization was observed with T308 receptors following histamine treatment. A mutant with a deletion of the 30 C-terminal amino acids, termed T329, was functional but was as potent as the WT in terms of cAMP production. Apart from being desensitized by histamine, the internalization of the receptor was indistinguishable from that of the WT. Internalization was observed in the T320 but not in T313 mutant, narrowing the region involved in internalization to that between Glu314 and Asn320 (ETSLRSN). Of these seven residues, either Thr315, Ser316, or both, were replaced with Ala. Thr315 and Ser316 are conserved among species. The mutation at Thr315 (but not that at Ser316) abolished internalization. Taken together, these results demonstrate that Thr315 is involved in agonist-induced internalization. Furthermore, the finding that T308 receptors were desensitized in the absence of internalization suggests that internalization and desensitization are meditated by independent mechanisms.

Amino Acid Sequence↗
PubMed1997

Protective and therapeutic immunity against leukemia induced by irradiated B7-1 (CD80)-transduced leukemic cells.

B7 molecules provide an important co-stimulatory signal for T cell receptor/CD3-mediated T cell activation via binding to their cognate receptors, CD28 and CTLA-4. We have introduced B7-1 (CD80) into M1 cells, spontaneously occurring mouse myelocytic leukemic cells, and assessed its potential to induce antitumor immunity to leukemia cells. Syngeneic, immunocompetent SL mice receiving two independent B7-1-transduced monoclonal sublines, M1-B7-1/F/clone F20 and M1-B7-1/F/clone F7, were rejected in 57% and 43% of SL mice, respectively. In vivo depletion of T cell subsets showed that both CD4+ and CD8 T cells were indispensable for the B7-1-dependent anti-leukemic immunity. Although a single exposure of irradiated monoclonal M1-B7/1/F cells was not fully effective, multiple exposures induced protective immunity against subsequent challenge with parental M1 cells. Furthermore, multiple vaccinations with irradiated monoclonal M1-B7-1/F/clone F7 cells could cure 67% of mice previously injected with a lethal number of M1 cells. These results emphasize that multiple exposures of irradiated B7-1-transduced myeloid leukemic cells can induce protective and therapeutic immunity against leukemia and that B7-1-mediated gene therapy may have therapeutic efficacy for patients with acute myelocytic leukemia.

Animals↗
PubMed1997

Effects of apo E deficiency on plasma lipid levels in mice lacking APOBEC-1.

Apolipoprotein (apo) B100 mRNA undergoes site specific C to U editing, generating a stop-translation codon of apo B48 in the small intestine. This reaction is catalyzed in an editosome which contains APOBEC-1, a catalytic subunit. To clarify the functional significance of the apo B mRNA editing in lipoprotein metabolism, we have generated APOBEC-1 knockout mice and double knockout mice which are deficient in both APOBEC-1 and apo E. The apo B mRNA editing activity was markedly reduced and complete elimination of apo B48 from the plasma was observed in APOBEC-1(-/-) mice. Plasma triglyceride levels significantly increased in the double knockout mice (APOBEC-1(-/-);apo E(-/-)) as compared to apo E(-/-) mice. These results suggest that APOBEC-1(-/-) mice are a valuable model for experiments designed to understand a role of apo B mRNA editing.

APOBEC-1 Deaminase↗
PubMed1997

Multiple exocytotic pathways in pancreatic beta cells.

Ca2+-dependent exocytotic pathways in mouse pancreatic beta cells were investigated using both capacitance measurement and amperometric detection of vesicular contents. Serotonin was preloaded into large dense-core vesicles for the amperometry. Exocytosis was induced by rapid elevation of cytosolic Ca2+ concentrations using caged-Ca2+ compounds. Capacitance measurement revealed two major components of exocytosis, and only the slow component was accompanied by amperometric events reflecting quantal serotonin secretion. Moreover, the fast and slow exocytoses induced the two forms of endocytosis that were reported to follow the exocytoses of small-clear and large dense-core vesicles, respectively. Interestingly, we recorded two types of responses of quantal events: in the type-1 response, most quantal events occurred with a delay of 0.2 s and were rapidly exhausted with a time constant of 1.7 s, while, in the type-2 response, quantal events occurred with a delay of 2.5 s and were sustained. This suggests the existence of two pathways or modes of the exocytosis involving large dense-core vesicles. Thus, we have revealed three exocytotic pathways with divergent fusion kinetics in beta cells, which provide a new basis for the understanding of the physiology and pathology of beta cells.

Animals↗
PubMed1997

Bcl-2 protein inhibits oxysterol-induced apoptosis through suppressing CPP32-mediated pathway.

Oxysterols are presumed to mediate cytotoxicity of oxidized LDL in atherosclerotic lesions. To elucidate its molecular mechanism, we established murine macrophage-like P388-D1 cells which over-express Bcl-2 protein by retrovirus-mediated gene transfer. Oxysterols (7-ketocholesterol, 25-hydroxycholesterol) induced nuclear condensation and oligonucleosomal DNA fragmentation, which were partially inhibited by Bcl-2 over-expression. Though CPP32 inhibitor suppressed the cell death in control cells, it showed no additive protection in the cells over-expressing Bcl-2. These findings indicate that oxysterols induce apoptosis via Bcl-2-inhibitable and -uninhibitable pathways, and the former depends on CPP32 activation.

Animals↗
PubMed1997

Redifferentiation of smooth muscle cells after coronary angioplasty determined via myosin heavy chain expression.

BACKGROUND: The pathophysiology of phenotypic modulation of smooth muscle cells (SMCs) involved in restenosis after angioplasty is not well understood. Smooth muscle myosin heavy chain (SM MHC) isoforms (SM1 and SM2) are specific markers for SMC differentiation. In particular, SM2 is useful as a marker of mature SMCs. SMemb is a nonmuscle myosin heavy chain (NM MHC) whose expression is upregulated in immature or activated SMC. METHODS AND RESULTS: To determine SMC phenotypes in neointimal tissues after percutaneous transluminal coronary angioplasty (PTCA), we performed immunohistochemistry on human coronary arteries with antibodies against alpha-SM actin, SM1, SM2, and SMemb. Tissues were obtained from six autopsied patients and from atherectomy specimens from 16 patients who had undergone PTCA. Medial SMCs were positive for alpha-actin, SM1, and SM2. Expression of SM1 and SM2 in the neointima varied with the time after intervention, whereas alpha-actin was constitutively expressed in all cases studied. Neointimal cells at 16 and 20 days after PTCA contained alpha-actin but little or no SM1 or SM2, indicating that these cells modulated their phenotype to the immature state. Neointimal SMCs recovered SM MHC expression, first SM1 and then SM2, by 6 months after PTCA. Increased expression of SMemb was found in the neointima but without apparent relationship to the time after PTCA. CONCLUSIONS: Neointimal SMCs show features of an undifferentiated state, indicated by altered expression of SM MHC, and undergo redifferentiation in a time-dependent manner. The expression of SM MHC isoforms provides insight into the biology of healing after angioplasty and furnishes useful tools for the understanding of the roles of differentiation and phenotypic modulation of SMCs in human vascular lesions.

Aged↗
PubMed1997

Selective expression of mRNA coding for the truncated form of erythropoietin receptor in hematopoietic cells and its decrease in patients with polycythemia vera.

The mRNA encoding full-length erythropoietin (EPO) receptor (EPOR-F) comprises exons I through VIII. Another membrane-bound EPOR (EPOR-T) isoform has a truncated cytoplasmic region and is encoded by the mRNA containing unspliced intron VII (EPOR-T mRNA). EPOR-T is believed to have a dominantly negative function against EPOR-F. We show that EPOR-T mRNA is markedly decreased in the blood cells of patients with polycythemia vera (PV). We also show that EPOR-T mRNA is not detected in erythroid/megakaryocytic leukemia cell lines, but is expressed in nonerythroid/nonmegakaryocytic lines, suggesting the presence of a cell type-specific system by which intron VII of the EPOR transcript is spliced. Deregulation of this splicing system in early hematopoietic progenitors possibly explains the profound decrease in EPOR-T mRNA and consequent pathophysiology of PV.

Cell Line↗
PubMed1997

The phosphatidylinositol 3' kinase pathway is required for the survival signal of leukocyte tyrosine kinase.

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase which belongs to the insulin receptor superfamily and is mainly expressed in pre-B lymphocytes and neuronal tissues. Recently, we demonstrated that LTK utilizes Shc and IRS-1 as two major substrates and while both equally activate the Ras pathway, only IRS-1 suppresses apoptosis of hematopoietic cells, suggesting the existence of another unidentified signaling pathway downstream of IRS-1, which is relevant to the anti-apoptotic activity. In the present study, we found that wortmannin, a specific inhibitor of phosphatidylinositol 3' (PI3)-kinase, abolished the survival effects of LTK. Although c-Cbl is found to be phosphorylated by LTK and therefore is a second candidate linking LTK with the PI3-kinase pathway along with IRS-1, we found that the p85 subunit of PI3 kinase directly binds to tyrosine 753 of LTK, which is located within a YXXM motif, a consensus binding amino acid sequence for the SH2 domain of p85, but fails to bind to IRS-1 or c-Cbl. Ba/F3 cells which stably express the EGF receptor-LTK chimeric receptor carrying a mutation at tyrosine 753 fell into apoptotic death even in the presence of EGF, indicating that the PI3 kinase pathway is required for the survival effects of LTK.

Androstadienes↗
PubMed1997

Oligomer formation of histamine H2 receptors expressed in Sf9 and COS7 cells.

A histamine H2 receptor, which had been mutated at its glycosylation site and tagged at its N-terminus with an HA tag (HA-H2 receptor), was expressed in Sf9 cells and COS7 cells. Immunoprecipitation and immunoblotting of HA-H2 receptors with alphaHA antibody revealed four bands of 31.5 +/- 2.5 kDa, 59.0 +/- 6.0 kDa, 80.5 +/- 4.5 kDa and 120 kDa. These bands were also detected by immunoblot using anti-H2 receptor serum (C-terminus). In addition, H2 receptors without the HA-tag coimmunoprecipitated with HA-tagged H2 receptors devoid of the 51 C-terminal amino acids, via immunoprecipitation with alphaHA antibody, when the two receptors were coexpressed. These results suggest that H2 receptors are present as receptor oligomers and that the C-terminal portion is not involved in the formation of these oligomers.

Animals↗
PubMed1997

Identification of PU.1 and Sp1 as essential transcriptional factors for the promoter activity of mouse tec gene.

Tec is a cytoplasmic protein-tyrosine kinase abundantly expressed in hematopoietic precursor cells. To investigate the mechanism regulating the expression of Tec molecule, we cloned and analysed 5' flanking region of mouse tec gene up to -2kb from the transcriptional initiation site. Luciferase assays using successive deletion mutants demonstrated that regions from -364 to -323 and from -122 to -63, which contain the consensus binding sequences for PU.1 (GGAA) and Sp1 (GGGCGG), respectively, are important for the transcriptional activity. Gel-shift and supershift assays revealed that PU.1 and Sp1 bind to the these regions through their consensus binding motifs. In addition, introduction of mutations into these motifs resulted in marked decrease in the promoter activity. These results indicate that PU.1 and Sp1 are essential for the transcriptional activity of the tec promoter and suggest that the cooperation of PU.1 and Sp1 plays a substantial role in the preferential expression of the Tec molecule in the hematopoietic lineages.

Animals↗
PubMed1997

Insulin receptor substrate (IRS)-2 is dephosphorylated more rapidly than IRS-1 via its association with phosphatidylinositol 3-kinase in skeletal muscle cells.

Insulin receptor substrate (IRS)-2 is structurally and functionally similar to IRS-1. Indeed, stimulation with insulin or insulin-like growth factor I led to the rapid tyrosine phosphorylation of both IRS-1 and IRS-2, which in turn activated phosphatidylinositol (PI) 3-kinase in L6 cells and rat skeletal muscle. However, IRS-2 was rapidly dephosphorylated (3-10 min after the addition of insulin/insulin-like growth factor I), whereas IRS-1 phosphorylation continued for at least 60 min. The time courses of the PI 3-kinase activity associated with IRS-1 and IRS-2 paralleled the tyrosine phosphorylation of these proteins. Preincubation with sodium orthovanadate, an inhibitor of protein tyrosine phosphatase, blocked the rapid dephosphorylation of IRS-2, suggesting the involvement of tyrosine phosphatase. The activation of PI 3-kinase apparently plays an important role in the rapid dephosphorylation of IRS-2, as IRS-2 dephosphorylation was inhibited markedly by suppressing PI 3-kinase activity with wortmannin or overexpression of the dominant negative p85 subunit of PI 3-kinase, which cannot bind the p110 catalytic subunit. In addition, platelet-derived growth factor stimulation prior to insulin stimulation decreased IRS-associated PI 3-kinase and significantly inhibited the dephosphorylation of IRS-2. Taken together, these observations suggest that IRS-2 plays a unique role in mediating the signals from the insulin receptor to downstream molecules and that this effect is more transient than that of IRS-1. Tyrosine phosphatase and IRS-associated PI 3-kinase activity thus contribute to the rapid dephosphorylation of IRS-2.

Androstadienes↗
PubMed1997

Suppression of neutral cholesterol ester hydrolase activity by antisense DNA of hormone-sensitive lipase.

In order to investigate the role of the hormone-sensitive lipase (HSL) gene in the activity of neutral cholesterol ester hydrolase (NCEH) from a molecular perspective, Chinese hamster ovary (CHO) cells were transfected with rat antisense hormone-sensitive lipase cDNA, and three different cell lines, designated as anti-HSL, were established. NCEH activity in anti-HSL cells was reduced to approximately 50% of that in control CHO cells. The concentration of cellular esterified cholesterol increased and the concentration of free cholesterol decreased in the anti-HSL cell lines. These results suggest that the HSL gene has a function of NCEH as well.

Animals↗
PubMed1997

Genetic polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR) as a risk factor for coronary artery disease.

BACKGROUND: Epidemiological studies have identified hyperhomocyst(e)inemia as an independent risk factor for coronary artery disease (CAD). Recently, the alanine/valine (A/V) polymorphism of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, one of the key enzymes catalyzing remethylation of homocysteine, has been reported. The VV genotype correlates with increased plasma homocyst(e)ine levels as a result of the reduced activity and increased thermolability of this enzyme. In this study, we examined the distribution of the MTHFR genotypes in Japanese men and the association between the VV genotype and CAD. METHODS AND RESULTS: The diagnoses of CAD of all the studied patients were confirmed by coronary angiography. The MTHFR genotype was analyzed by PCR followed by HinfI digestion. In 778 healthy male subjects, the frequency of the V allele was 0.33, comparable to that in a French Canadian population. In 362 patients with CAD, the VV genotype was significantly more frequent than in control subjects (16% versus 10%, P=.0067). The association of the VV genotype with CAD was further increased in patients with > or = 99% stenotic lesions (18%, P=.0010), whereas no significant association with the VV genotype was observed in patients without a > or = 99% stenosis. When the genotype frequency was compared among patients with different numbers of stenotic coronary arteries, the frequency of the VV genotype was significantly higher in patients with triple-vessel disease (26%) than in patients with single- or double-vessel disease (15% and 14%, respectively). CONCLUSIONS: The VV genotype of MTHFR was also common in the Japanese population and was significantly associated with CAD. The frequency of this genotype in particular was correlated with the severity of disease. The VV genotype associated with a predisposition to increased plasma homocyst(e)ine levels may represent a genetic risk factor for CAD.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗
PubMed1997

Purification and molecular cloning of SH2- and SH3-containing inositol polyphosphate-5-phosphatase, which is involved in the signaling pathway of granulocyte-macrophage colony-stimulating factor, erythropoietin, and Bcr-Abl.

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with Shc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important role in the signaling pathway triggered by GM-CSF and in the transformation of hematopoietic cells caused by Bcr-Abl.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Antisense repression of proto-oncogene c-Cbl enhances activation of the JAK-STAT pathway but not the ras pathway in epidermal growth factor receptor signaling.

Many growth factors including epidermal growth factor (EGF) induce tyrosine phosphorylation of the c-Cbl proto-oncogene product, whose function, however, remains unclear. Recently, Sli-1, a Caenorhabditis elegans homologue of c-Cbl, was found to be a negative regulator of let-23-mediated vulval induction pathway, suggesting that c-Cbl may negatively regulate EGF receptor (EGFR)-mediated signaling. In this study, by an antisense RNA approach, we examined the effects of expression level of c-Cbl on EGFR signaling and showed that overexpression of c-Cbl reduces and antisense repression of c-Cbl enhances autophosphorylation of EGF receptors and activation of the JAK-STAT pathway. However, in contrast to the Sli-1 protein, the expressed amount of c-Cbl does not affect activation of the Ras pathway, suggesting that the EGFR-mediated signaling pathways are differently regulated by c-Cbl among nematodes and mammals.

3T3 Cells↗