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Biomedical subjects

Y Yazaki

Publications and source records attributed to Y Yazaki.

At least 343 records · Page 19Linked to original sources
PubMed1997

Roles of PI 3-kinase and Ras on insulin-stimulated glucose transport in 3T3-L1 adipocytes.

The dominant negative p85alpha regulatory subunit (delta p85alpha) of phosphatidylinositol (PI) 3-kinase or dominant negative Ras (N17Ras) was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Functional expression of delta p85alpha and N17Ras was confirmed by marked inhibition of insulin-stimulated PI 3-kinase activity and mitogen-activated protein kinase activity, respectively. N17Ras expression did not affect glucose transport activity, whereas delta p85alpha expression inhibited insulin-stimulated glucose transport with impairment of GLUT-4 translocation, although inhibition of glucose transport activity was less remarkable than that of PI 3-kinase activity in delta p85alpha-expressing cells. Thus the Ras signaling pathway does not play a major part in either translocation or intrinsic activity of glucose transporters, but PI 3-kinase activation, via phosphotyrosyl proteins and heterodimeric PI 3-kinase, plays a pivotal role in insulin-stimulated glucose transport. However, a discrepancy was observed between PI 3-kinase activity and glucose transport activity, suggesting a possibility that a different pathway(s) is involved in insulin-stimulated intrinsic activity of glucose transporters.

3T3 Cells↗
PubMed1997

Skipping of exon 14 and possible instability of both the mRNA and the resultant truncated protein underlie a common cholesteryl ester transfer protein deficiency in Japan.

Among the Japanese population, a G-to-A mutation at the beginning of intron 14 of the human cholesteryl ester transfer protein (CETP) gene is a frequent cause of CETP deficiency characterized by markedly increased HDL cholesterol. The resulting abnormalities responsible for null CETP deficiency were studied in detail. The CETP mRNA transcripts amplified by polymerase chain reaction from the monocyte-derived macrophages of two homozygous patients were both found to be normal except for the whole deletion of exon 14. The deletion causes a shift of reading frame and introduces a premature termination codon downstream. Examination of the macrophage RNA from heterozygotes suggested the increased instability of the abnormal mRNA in the cytoplasm, because the amount of the aberrant transcript was nearly one third that of a normal transcript in the cytoplasm, while they were equal in the nucleus. Although this indicated the synthesis of a mutant CETP that lacks about 15% at its carboxy terminus, immunoblot analysis demonstrated that the abnormal CETP was virtually absent in both the media and cell lysates of transfected COS-1 cells, which massively expressed the mutant CETP mRNA. These results elucidate the primary abnormality due to the common CETP splicing mutation to be the exon skipping of mRNA, which decreases the level of mRNA and produces a truncated protein that should be rapidly degraded intracellularly.

Alternative Splicing↗
PubMed1997

Angiotensin II stimulates c-Jun NH2-terminal kinase in cultured cardiac myocytes of neonatal rats.

Many lines of evidence have suggested that angiotensin II (Ang II)plays an important role in cardiac hypertrophy. Ang II not only increases protein synthesis but also induces the reprogramming of gene expression in cultured cardiac myocytes. In the present study, to elucidate the mechanism by which Ang II regulates gene expression in cardiac myocytes, we examined whether Ang II activates c-Jun NH2-terminal kinase (JNK), which is a member of the mitogen-activated protein kinase family and activates the transcription factor, activator protein-1 (AP-1). The activity of JNK increased 5 minutes after the addition of Ang II, peaked at 20 minutes, and gradually decreased thereafter. Examination of the Ang II dose-response relation revealed detectable JNK activation at 10(-9) mol/L and maximal activation at 10(-6) mol/L. Ang II activated JNK through the AT1 receptor, and the activation was attenuated by the downregulation of protein kinase C or the chelation of intracellular Ca2+. Although the addition of either Ca2+ ionophore or phorbol ester resulted in little or no activation of JNK, simultaneous addition of both Ca2+ ionophore and phorbol ester markedly activated JNK. Slight expressions of the c-jun gene were observed in unstimulated cardiac myocytes, and Ang II increased expressions of the c-jun gene as well as the c-fos gene. Ang II increased transcription of the endothelin-1 gene through the AP-1 binding site. In conclusion, Ang II may activate JNK in cultured cardiac myocytes through an increase in intracellular Ca2+ and activation of protein kinase C, and the activated JNK may regulate gene expression by activating AP-1 during Ang II-induced cardiac hypertrophy.

Angiotensin II↗
PubMed1997

Roles of insulin receptor substrate-1 and Shc on insulin-like growth factor I receptor signaling in early passages of cultured human fibroblasts.

Insulin-like growth factor-I (IGF-I) improves glucose metabolism and growth in patients with leprechaunism. We investigated signal transduction through IGF-I receptor in comparison with epidermal growth factor (EGF) receptor in early passages of cultured skin fibroblasts from a normal subject and a patient with leprechaunism whose insulin receptor tyrosine kinase was almost nonexistent. Insulin receptor substrate-1 (IRS-1) became tyrosine-phosphorylated and bound growth factor receptor-bound protein 2 (GRB2) quickly by IGF-I. The association of Shc with GRB2 by IGF-I was detected by immunoblot with anti-Shc antibody but was hardly visible with antiphosphotyrosine antibody, which was in marked contrast to efficient tyrosine phosphorylation of Shc by EGF. However, the potency of IGF-I for DNA synthesis was far stronger than EGF, which was not parallel with the potency of these growth factors to activate Shc or MAP kinase. Rather, phosphatidylinositol (PI) 3-kinase activity, which was activated by IGF-I about 5- to 10-fold more strongly than EGF, appeared to correlate with mitogenesis. Signal transduction pathways following IGF-I receptor or EGF receptor activation were indistinguishable between the normal subject and the patient. Our results strongly suggest that in human skin fibroblasts, which represent a more physiological cell culture: 1) IRS-1, rather than Shc, is the major tyrosine-phosphorylated protein binding GRB2 in initial phase of IGF-I signaling; 2) mitogenic potency of receptor tyrosine kinases such as IGF-I receptor and EGF receptor may not be determined solely by the amount of Shc-GRB2 complex or the activity of MAP kinase; and 3) in contrast to previous reports, IGF-I and EGF receptor signalings are not defective in leprechaunism.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Targeting of GLUT1-GLUT5 chimeric proteins in the polarized cell line Caco-2.

Caco-2, a human differentiated intestinal epithelial cell line, is a promising model for investigating the mechanism of polarized targeting of apical and basolateral membrane proteins. We stably transfected rat GLUT5 cDNA and rabbit GLUT1 cDNA into Caco-2 cells with an expression vector. Immunohistochemical study revealed that the GLUT5 protein expressed was localized at apical membranes and that the GLUT1 expressed was present primarily in the basolateral membranes of cells grown on permeable support. Next, to investigate the domain responsible for determining apical vs. basolateral sorting in glucose transporters, we prepared several GLUT1-GLUT5 chimeric cDNAs and transfected them into Caco-2 cells. A GLUT1 [N terminus approximately sixth transmembrane domain (TM6)]-GLUT5 [intracellular loop (IL) approximately C terminus] chimera was observed exclusively at the apical membrane, while GLUT1 (N terminus approximately IL)-GLUT5 (TM7 approximately C terminus) and GLUT1 (N terminus approximately TM12)-GLUT5 (C-terminal domain) chimeras were observed mainly at the basolateral membrane, a localization similar to that of GLUT1. Moreover, using a recombinant adenovirus expression system, we expressed a GLUT5 (N terminus approximately TM6)-GLUT1(IL)-GLUT5(TM7 approximately C-terminus) chimera, which was observed at the basolateral membrane. Based on these results, the C-terminal domain does not determine isoform-specific targeting of GLUT1 and GLUT5. Rather, it is the intracellular loop in glucose transporters that appears to play a pivotal role in apical-basolateral sorting signals in Caco-2 cells.

Animals↗
PubMed1997

Perforin-secreting killer cell infiltration in the aortic tissue of patients with atherosclerotic aortic aneurysm.

Cell-mediated immunity has been implicated in the pathogenesis of vascular cell injury in patients with atherosclerotic aortic aneurysms. To clarify the immunologic mechanisms involved, we examined the expression of a cytolytic factor, perforin, in infiltrating cells from aortic tissue samples taken from 6 patients with atherosclerotic aortic aneurysms. Immunohistochemical studies showed that the infiltrating cells consisted mainly of macrophages, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and T helper cells, and that perforin was expressed in NK cells and CTLs. Immunoelectron microscopic studies demonstrated that the infiltrating cells released massive amounts of perforin directly on to the surface of arterial vascular cells. These findings provide the first direct evidence that some of the infiltrating cells in the aortic tissue consist of killer cells, and strongly suggest that these killer cells, especially NK cells and CTLs, may play a critical role in the vascular cell injury caused by atherosclerotic aortic aneurysm by releasing perforin.

Aged↗
PubMed1997

Germ-line contribution of embryonic stem cells in chimeric mice: influence of karyotype and in vitro differentiation ability.

The effect of the karyotype and the ability to differentiate in vitro upon germ-line transmission by A3-1 embryonic stem (ES) cells in chimeric mice were examined. Germ-line transmission was confirmed in ES cells exhibiting 38% and more of the normal karyotype, but no chimeric mice and/or germ-line transmitters were observed regardless of the karyotype when the cystic embryoid body (CEB) was formed on day 8 and later in the suspension culture. Germ-line transmission of the ES cells was not significantly influenced by formation of the simple embryoid body (SEB). Germ-line transmitters were preferentially observed in chimeras when the ES cell contribution to coat color was markedly increased, but this contribution to coat color varied regardless of the karyotype or in vitro differentiation ability. These results suggest that A3-1 ES cells which exhibit CEB at 7 days after suspension culture and approximately 40% of normal karyotype are capable of germ-line transmission in chimeric mice.

Animals↗
PubMed1997

Fulminant septicemic syndrome of Bacillus cereus in a leukemic patient.

We report a rapidly fatal Bacillus cereus septicemia in a leukemic patient receiving remission-induction therapy. Symptoms resembling food poisoning and fever preceded coma accompanied by neurologic abnormalities. Autopsy revealed necrotizing leptomeningitis with subarachnoid hemorrhage and coagulation necrosis of the liver with bacterial infiltration. These clinicopathologic findings were closely similar to those of reported cases. Because of a rapidly fatal clinical course, suspicion of this syndrome early in the course is important to determine an appropriate treatment. Therefore, we propose that this type of septicemia should be termed as fulminant septicemic syndrome of Bacillus cereus.

Bacillus cereus↗
PubMed1997

The multiple roles of macrophage scavenger receptors (MSR) in vivo: resistance to atherosclerosis and susceptibility to infection in MSR knockout mice.

Both type I and type II MSRs are integral membrane proteins containing a collagenous domain and elicit an extraordinarily wide range of ligand binding capability. They were found during the search for the molecule(s) responsible for the accumulation of modified LDL during atherogenesis. However, all prior the evidence relating to their physiological and pathophysiological roles in vivo had been indirect. Targeted disruption of the MSR gene results in a reduction in the size of atherosclerotic lesions in an apo E deficient animal. Macrophages from MSR deficient mice exhibit a marked decrease in modified LDL uptake in vitro, whereas modified LDL clearance from plasma remains normal, suggesting that there are alternative mechanisms for the uptake of modified LDL from the circulation. In addition, MSR knockout mice are more susceptible to L. monocytogenes and HSV-1 infection, indicating a role for MSR in host defense against various pathogens.

Animals↗
PubMed1997

An acute myeloid leukemia gene, AML1, regulates transcriptional activation and hemopoietic myeloid cell differentiation antagonistically by two alternative spliced forms.

The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) and (3;21)(q26;q22) translocations associated with myelogenous leukemias and encodes a DNA-binding protein. From AML1 gene, two representative forms of proteins, AML1a and AML1b, are produced by an alternative splicing. Both forms have DNA-binding domain, but AML1a lacks a putative transcriptional activation domain which AML1b has. Here we demonstrate that AML1a, which solely has no effects as a transcriptional regulator, dominantly suppresses transcriptional activation by AML1b, and that AML1a exhibits the higher affinity for DNA-binding than AML1b. Furthermore a dominant negative form of AML1, AML1a, totally suppressed granulocytic differentiation otherwise induced by granulocyte colony-stimulating factor when AML1a was overexpressed in 32Dc13 murine myeloid cells. Such differentiation block by AML1a was canceled by the concomitant overexpression of AML1b. These data strongly suggest that a transcriptionally active form of AML1 is essential for the myeloid cell differentiation. In addition, we observed an altered expression level of AML1 along with the myeloid differentiation in several hemopoietic cell lines. In these cases, at least, the AML1 expression level is a potential regulator for myeloid cell differentiation.

Alternative Splicing↗
PubMed1997

Recent progress in molecular mechanisms of leukemogenesis: the cyclin-dependent kinase 4-inhibitor gene in human leukemias.

In order to clarify the significance of p16 gene (CDKN2) inactivation and its disease specificity among hematopoietic tumors, configurations of the p16 gene as well as those of the adjacent p15 and interferon alpha (IFN alpha) genes were examined in primary hematopoietic tumors. Loss of the p16 gene is frequent in and highly specific to lymphoid tumors among hematopoietic tumors. Gene deletions but not minute mutations should be the predominant mechanism of p16 gene inactivation in these types of tumors. The p16 gene is most frequently deleted among the p16, p15 and IFN alpha genes and thus should be the target of deletions in this locus. Deletions of the p16 gene were frequently observed in tumors carrying chromosome 9p abnormalities while a significant number of cases showed loss of the p16 gene without chromosome 9p abnormalities. So far inactivation of p53 and Rb tumor suppressors have also been found in lymphoid tumors. In our study, we detected homozygous deletions of p16 gene in 20%, loss of Rb protein in 28%, and p53 gene alterations in 8% of lymphoid tumors. Notably, 44% of lymphoid tumors showed inactivation of at least one of the three tumor suppressors, suggesting these tumor suppressors are important for lymphoid tumorigenesis. Inactivations of these tumor suppressors should independently occur in development of lymphoid tumors.

Carrier Proteins↗
PubMed1997

The signal transduction through Grb2/Ash in hematopoietic cells.

Grb2/Ash is composed of one SH2 and two SH3 domains and functions as an adapter linking tyrosine-kinase receptors and Ras in fibroblasts. The SH2 domain binds to tyrosine-phosphorylated proteins and the SH3 domain binds to protein containing proline-rich regions. However, the mechanisms of signal transduction through Grb2/Ash in hematopoietic cells are still unclear. By means of the binding experiments using the GST fusion protein including the full length Grb2/Ash, we have found that Shc and unidentified 130-kDa and 135-kDa proteins are associated with Grb2/Ash and that they are tyrosine-phosphorylated by treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) in a human leukemia cell line UT-7. We have purified the 130-kDa protein (pp 130) using GST-GRB2/Ash affinity column. The amino-acid sequence analysis showed that the pp130 was identical to the human c-cbl proto-oncogene product (c-Cbl). c-Cbl constitutively binds to the SH3 domain of Grb2/Ash both in vitro and in vivo but not to the SH2 domain of Grb2/Ash. Moreover, c-Cbl (pp 130) becomes tyrosine-phosphorylated rapidly and transiently depending on GM-CSF and EPO stimulation. However, we could not find the homologous regions with guanine nucleotide exchange factors or GTPase-activating proteins in the c-cbl gene. These findings strongly suggest that c-Cbl is implicated in the signal transduction of GM-CSF and EPO in hematopoietic cells, and c-Cbl and Grb2/Ash might also transduce a signal that is different from the signal leading to Ras regulation. Recently, we have shown that the proto-oncogene vav product (Vav) is also tyrosine-phosphorylated by treatment with GM-CSF and EPO and is constitutively associated with the SH3 domain of Grb2/Ash in UT-7. Another guanine nucleotide exchange factor Sos is also associated with Grb2/Ash in UT-7. It has been reported that Vav has guanine nucleotide exchange activity and activates Ras in vitro and in vivo. These data suggest that tyrosine kinases, the adapter Grb2/Ash, and the guanine nucleotide exchange factor Vav and Sos are members of a signaling pathway leading to Ras activation in hematopoietic cells.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Of the GATA-binding proteins, only GATA-4 selectively regulates the human IL-5 gene promoter in IL-5 producing cells which express multiple GATA-binding proteins.

Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region, within the human IL-5 gene promoter, that regulates IL-5 gene transcription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of these family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and PMA/A23187 stimulation are necessary for the IL-5 promoter activation. The requirement of another regulatory element called CLE0, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNA of three GATA-binding proteins, hGATA-2, hGATA-3 and hGATA-4, and each of them has a potential to bind to the consensus (A/T)GATA(G/ A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms specific DNA-protein complex with the -70 GATA site. The electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity to the -70 GATA site among the three GATA-binding proteins. When the transactivation ability was compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed.

Base Sequence↗
PubMed1997

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

B7 molecules provide an important costimulatory 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-occurred mouse myelocytic leukemic cells and assessed its potential in the induction immunity to leukemia cells. Syngeneic, immunocompetent SL mice receiving polyclonal B7-1-transduced M1 cells showed prolonged survival than control mice. Two independent B7-1-transduced monoclonal sublines, M1-B7-1+ (F20) and M1-B7-1+ (F7), were rejected in 100% an 50% 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 to irradiated monoclonal M1-B7-1+ cells were not fully effective, multiple exposures induced protective immunity against subsequent challenge with M1 cells. Furthermore, hyperimmunization with irradiated monoclonal M1-B7-1+ (F7) cells could partly cure mice previously injected with a lethal number of M1 cells. Although other groups have demonstrated that live, proliferating B7-1-transduced leukemic cells can improve antitumor immunity, this is the first report which shows that irradiated B7-1-transduced myeloid leukemic cells can induce protective and therapeutic immunity against leukemia.

Animals↗
PubMed1997

[Gene Polymorphism of 5, 10-methylenetetrahydrofolate reductase as a coronary risk factor].

Hyperhomocysteinemia has been identified as a possible risk factor for coronary artery disease. The association of the alanine/valine (A/V) polymorphism of 5, 10-methylenetetrahydrofolate reductase (MTHFR), one of the key enzymes catalyzing re-methylation of homocysteine, with coronary artery disease was examined in 362 Japanese males with a diagnosis of coronary artery disease confirmed with coronary angiography. The A/V polymorphism was analyzed with PCR followed by Hinf I digestion. The screening of 778 male volunteer controls revealed that the frequency of V allele in Japanese was 0.33, comparable to that in the French Canadian population. The VV genotype, which correlates with increased plasma homocysteine levels due to reduced activity and increased thermolability of this enzyme, was significantly more frequent in patients with coronary artery disease (15.7%, n = 362) than in controls (10.2%, n = 778; p = 0.0067). The association of the VV genotype with coronary artery disease was further increased in patients with > or = 99% stenotic lesion (p = 0.0010). In these patients, 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). The fasting plasma homocysteine levels in VV subjects were higher than those in AV or AA subjects. The VV genotype of MTHFR associated with increased plasma homocysteine levels may represent an important genetic risk factor for coronary artery disease, especially with the occurrence of myocardial infarction.

Adult↗
PubMed1997

[Molecular mechanism of cardiac hypertrophy and dysfunction].

We examined the signal transduction pathway for the development of cardiac hypertrophy induced by high blood pressure. The activities of Raf-1 kinase (Raf-1), mitogen-activated protein kinase kinase (MAPKK), MAP kinases (MAPKs) and 90-kDa ribosomal S6 kinase (p90rsk) was examined by passively stretching neonatal rat cardiomyocytes in vitro. Mechanical stretch activated these protein kinases transiently and sequentially: the maximal activation of Raf-1, MAPKK, MAPKs and p90rsk was observed at 2 minutes, 5 minutes, 8 minutes and 10 approximately 30 minutes, respectively. Both angiotensin II (AngII) and endothelin-1 (ET-1) were constitutively secreted from cultured cardiomyocytes, and a significant increase in the concentration was recognized in the culture medium of cardiomyocytes within 10 minutes after stretch. ET-1 mRNA levels were also increased in cardiomyocytes at 30 minutes after stretch. Moreover, ET-1 and AngII synergistically activated Raf-1 and MAPKs in cultured cardiomyocytes. In conclusion, mechanical stretch stimulates secretion and production of AngII and ET-1 in cultured cardiomyocytes, and both vasoconstrictive peptides may play an important role in mechanical stress (high blood pressure)-induced cardiac hypertrophy.

Angiotensin II↗