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Biomedical subjects

Y Yazaki

Publications and source records attributed to Y Yazaki.

At least 451 records · Page 25Linked to original sources
PubMed1996

Abnormal expression of Evi-1 gene in human leukemias.

The human Evi-1 gene located on chromosome 3q26, encodes a zinc finger protein that functions as a transcription factor. It was frequently overexpressed in leukemias having 3q26 abnormalities such as t(3;3)(q21;q26) and inv(3)(q21 q26), and subjected to structural alteration in t(3;21)(q26;q22). In addition, recent studies indicated that several cases of leukemias without 3q26 abnormalities also expressed Evi-1 gene. In this study we present another case of structural alteration of Evi-1 gene in a case of inv(3)(q21 q26), in which Evi-1 was truncated and a shorter form of Evi-1 protein was expressed upon rearrangement of the gene. We also studied expression of the Evi-1 gene in a variety of leukemias by northern blot analysis. Evi-1 was overexpressed not only in leukemias with 3q26 abnormalities, but, in those without 3q26 abnormalities, especially in blast crisis of CML. Our result also supports an idea that Evi-1 is a relevant oncogene whose overexpression or structural changes might play a crucial role in development of human leukemias.

Adult↗
PubMed1995

Adult-onset spinocerebellar dysfunction caused by a mutation in the gene for the alpha-tocopherol-transfer protein.

BACKGROUND: Patients with isolated vitamin E deficiency have an impaired ability to incorporate alpha-tocopherol into lipoproteins in the liver and usually have symptoms and signs of spinocerebellar dysfunction before adolescence. Accumulated evidence suggests that the alpha-tocopherol-transfer protein, which is presumed to function in the intracellular transport of alpha-tocopherol, is abnormal in these patients. METHODS: We studied a patient from an isolated Japanese island who began to have ataxia, dysarthria, and sensory disturbances in the sixth decade of life. His serum vitamin E concentration was low (1.2 micrograms per milliliter [2.8 mumol per liter]). Exons of his gene for the alpha-tocopherol-transfer protein were analyzed by DNA sequencing. We also screened an additional 801 inhabitants of the island for the mutation. Both the normal and mutant alpha-to-copherol-transfer proteins were expressed in COS-7 cells and studied by immunoblot analysis and assay for alpha-tocopherol-transfer activity. RESULTS: The patient was homozygous for a point mutation that replaces histidine (CAT) with glutamine (CAG) at position 101 of the gene for the alpha-tocopherol-transfer protein. When expressed in COS-7 cells, the missense mutation produced a functionally defective alpha-tocopherol-transfer protein with approximately 11 percent of the transfer activity of the wild-type protein. Of the 801 island inhabitants examined, 21 were heterozygous for the His101Gln mutation. In all affected subjects, including the patient, this mutation cosegregated with an intron-sequence polymorphism. The heterozygotes were phenotypically normal and had serum vitamin E concentrations that were on average 25 percent lower than those of normal subjects (mean [+/- SD], 7.5 +/- 2.2 vs. 10.1 +/- 2.8 micrograms per milliliter [17.4 +/- 5.1 vs. 23.4 +/- 6.5 mumol per liter]; P = 0.002). CONCLUSIONS: alpha-Tocopherol-transfer protein is a determinant of serum vitamin E concentrations. An abnormality in this protein is a cause of spinocerebellar dysfunction.

Adult↗
PubMed1995

Molecular cloning of the human glucose-regulated protein ERp57/GRP58, a thiol-dependent reductase. Identification of its secretory form and inducible expression by the oncogenic transformation.

Recently it was shown that putative phospholipase C-alpha cDNA does not code for an isotype of the phospholipase C superfamily but for one of the glucose-regulated proteins (GRPs), ERp57/GRP58. We have isolated human ERp57/GRP58 cDNA from human placenta. Sequence analysis showed that ERp57/GRP58 has two Trp-Cys-Gly-His-Cys-Lys motifs completely conserved among the mammals. Bacterially expressed recombinant ERp57/GRP58 protein contained a thiol-dependent reductase activity which was completely abolished when Ser residues were substituted for Cys residues in both of the two motifs. Furthermore, we have identified a soluble form of ERp57/GRP58 by Western blotting and biosynthetic labeling. In v-onc transformants of normal rat kidney cells, the expression level of ERp57/GRP58 was elevated at the protein level. In NIH3T3 cells transformed with v-src, activated c-src (Y527F) or c-src, the expression level of ERp57/GRP58 was upregulated in proportion to their transforming abilities. These results indicate that a soluble form of ERp57/GRP58 exists and that this protein may control both extracellular and intracellular redox activities through its thiol-dependent reductase activity. Moreover, it is likely that ERp57/GRP58 is involved in the oncogenic transformation.

3T3 Cells↗
PubMed1995

TPO/c-mpl ligand induces tyrosine phosphorylation of multiple cellular proteins including proto-oncogene products, Vav and c-Cbl, and Ras signaling molecules.

Thrombopoietin (TPO)/c-mpl ligand is a hematopoietic growth factor that stimulates proliferation and maturation of megakaryocytes. To analyze the signaling pathway downstream of the c-mpl product, we used a human megakaryoblastic cell line, Mo7e, that has been proved to be responsive to TPO in terms of DNA synthesis. In this study, we found that TPO treatment resulted in tyrosine phosphorylation of Jak-2 kinase. Moreover, it was revealed that several functional molecules involved in the Ras signaling pathway, Shc and Sos, were phosphorylated by treatment with TPO. Finally, tyrosine phosphorylation of the proto-oncogene products, Vav and c-Cbl, has been proved to be induced by TPO. These results suggest that TPO could activate several signaling pathways including the Jak/Stat pathway, the Ras pathway and possibly another pathway involving the c-Cbl proto-oncogene product.

Cell Cycle Proteins↗
PubMed1995

Cloning and structural analysis of the mouse apolipoprotein B mRNA editing protein gene.

Mouse apolipoprotein B mRNA editing protein gene has been isolated and characterized. The amino acid sequences deduced from the exonic sequences of the gene showed 90, 70 and 68% identity to those of rat, human and rabbit, respectively. RNA blotting experiments demonstrated high expression in small intestine especially in the jejunum as a major 2.4 kb mRNA species. mRNA in other tissues including the liver is smaller in amounts and larger in size (3.1 kb) than the intestinal mRNA. The gene spans 12 kb and contains 5 exons and 4 introns. Sequencing of the 5'-flanking region revealed that a canonical TATA box is absent and the consensus recognition sequences for several transcription factors have been identified.

APOBEC-1 Deaminase↗
PubMed1995

Inhibition of pancreatic beta-cell glucokinase by antisense RNA expression in transgenic mice: mouse strain-dependent alteration of glucose tolerance.

We have generated transgenic mice, in either C57BL/6 or C3H background, expressing antisense glucokinase mRNA in beta-cells. The glucose phosphorylating activity at 60 mM glucose in transgenic islets was significantly lower than that in controls, and the insulin secretory response to glucose was lower in transgenic islets than in those of controls in both strains. Following i.p. glucose challenge, higher blood glucose levels were observed in transgenic mice than in controls in the C57BL/6 but not the C3H background. These data suggest that a beta-cell secretory defect, in combination with other undefined genetic factors, causes impaired glucose homeostasis in mice.

Animals↗
PubMed1995

A novel immunoassay of smooth muscle myosin heavy chain in serum.

We have developed a double monoclonal sandwich enzyme immunoassay to measure smooth muscle myosin heavy chain (MHC). Analytical performance of the assay showed reliable detection of smooth muscle MHC in human sera. The mean of the smooth muscle MHC level in normal human sera was 0.9 +/- 0.9 ng/ml. In sera of patients with aortic dissection, the smooth muscle MHC level sharply elevated at the onset and rapidly decreased to normal levels. Immunoassay of smooth muscle MHC in serum is a promising method for biochemical diagnosis of smooth muscle disorders.

Aortic Dissection↗
PubMed1995

The AML1/Evi-1 fusion protein in the t(3;21) translocation exhibits transforming activity on Rat1 fibroblasts with dependence on the Evi-1 sequence.

The t(3;21) (q26;q22) chromosomal translocation associated with blastic crisis of chronic myelogenous leukemia (CML) results in the formation of a chimeric protein fusing the amino-terminal DNA-binding domain encoded by the AML1 gene to the carboxyl-terminal-encoding portion of the Evi-1 gene. In order to evaluate transforming activity of this protein, AML1/Evi-1 was introduced into Rat1 fibroblasts. Cells expressing the fusion product formed macroscopic colonies in soft agar, indicating that AML1/Evi-1 is a transforming gene. It was also demonstrated that introduction of AML1/Evi-1 into the Rat1 clones harboring BCR/ABL also conferred enhanced capacity for anchorage independent growth. Analyses of deletion mutants of AML1/Evi-1 revealed that removal of the second zinc finger domain within the Evi-1 sequence totally abrogated the ability of AML1/Evi-1 to transform Rat1 cells. We showed that the transforming effect is correlated with the AP-1 activation induced by AML1/Evi-1. Furthermore, we demonstrated that c-jun is transcriptionally activated in Rat1 cells transformed by AML1/Evi-1, suggesting that c-jun expression is under control of AML1/Evi-1. These results indicate that the oncogenic effect of the t(3;21) translocation is caused by the generation of a chimeric transcriptional factor and that AML1/Evi-1 could perform a pivotal role in leukemic progression of CML.

3T3 Cells↗
PubMed1995

Structural and functional analysis of the canine histamine H2 receptor by site-directed mutagenesis: N-glycosylation is not vital for its action.

G-protein-coupled receptors generally share a similar structure containing seven membrane-spanning domains and extracellular site(s) for N-glycosylation. The histamine H2 receptor is a member of the family of G-protein-coupled receptors, and has three extracellular potential sites for N-glycosylation (Asn-4, Asn-162 and Asn-168). To date, however, no information has been presented regarding N-glycosylation of the H2 receptor. To investigate the presence, location and functional roles of N-glycosylation of the H2 receptor, site-directed mutagenesis was performed to eliminate the potential site(s) for N-glycosylation singly and collectively. The wild-type and mutated H2 receptors were expressed stably in Chinese hamster ovary (CHO) cells or transiently in COS7 cells. Immunoblotting of the wild-type and mutated H2 receptors with an antiserum directed against the C-terminus of the H2 receptor showed that mutation at Asn-162, but not at Asn-168, resulted in a substantial decrease in the molecular mass. A mutation at Asn-4 led to a further decrease in the molecular mass. Tunicamycin treatment of the transfected cells yielded a sharp band with a molecular mass identical to that of the mutant devoid of all three potential sites for N-glycosylation. These findings indicate that the H2 receptor is N-glycosylated, and that N-glycosylation takes place mainly at two sites, Asn-4 and Asn-162. Neither the affinity for tiotidine nor that for histamine was affected by the mutagenesis. Immunocytochemistry and tiotidine binding showed that the mutated receptors were exclusively distributed on the cell surface in a fashion similar to that of the wild-type. In addition, the glycosylation-defective receptor was capable of activating adenylate cyclase and elevating the intracellular Ca2+ concentration in response to histamine in stable CHO cell lines. Thus N-glycosylation of the H2 receptor is not required for cell surface localization, ligand binding or functional coupling to G-protein(s).

1-Methyl-3-isobutylxanthine↗
PubMed1995

An epidermal growth factor receptor-leukocyte tyrosine kinase chimeric receptor generates ligand-dependent growth signals through the Ras signaling pathway.

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase that belongs to the insulin receptor family. LTK is mainly expressed in pre B cells and brain. Previously we cloned the full-length cDNA of human LTK, but no ligands have so far been identified, and hence, very little is known about the physiological role of LTK. To analyze the function of the LTK kinase, we constructed chimeric receptors composed of the extracellular domain of epidermal growth factor receptor and the transmembrane and the cytoplasmic domains of LTK and established cell lines that stably express these chimeric molecules. When cultured in medium containing EGF, growth of these cell lines was stimulated, and these fusion proteins became autophosphorylated and associated with Shc in vivo in a ligand-dependent manner. By treatment with EGF, Shc was associated with the Grb2/Ash-Sos complex. Our analyses demonstrate that LTK associates with Grb2/Ash through an internal adaptor, Shc, depending on a ligand stimulation. The LTK binding site for Shc was tyrosine 862 at the carboxyl-terminal domain and to a lesser extent tyrosine 485 at the juxtamembrane domain. Both of them are located in NP/AXY motif which is consistent with binding sites for Shc. These findings demonstrate that LTK can activate the Ras pathway in a ligand-dependent manner and that at least one of the functions of this kinase is involved in the cell growth.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Stretching mesangial cells stimulates tyrosine phosphorylation of focal adhesion kinase pp125FAK.

Mechanical loading of cultured mesangial cells has been shown to induce both increased production of matrix proteins and cell proliferation. This in vitro phenomenon has been implicated in the progression of glomerular sclerosis observed in a variety of glomerular diseases. However, it is not yet known how cells sense mechanical stress and transfer this sense into biochemical and biological events. In this study, we show that mesangial stretching rapidly stimulates tyrosine phosphorylation of non-receptor tyrosine kinase pp125FAK. Since pp125FAK is a major constituent of focal adhesions, our findings suggest that the focal adhesion may be a site where mechanical forces are translated into biochemical events, and that pp125FAK may play an important role in this signaling cascade.

Animals↗
PubMed1995

Overexpression of human lipoprotein lipase increases hormone-sensitive lipase activity in adipose tissue of mice.

An elevation of lipoprotein lipase (LPL) activity in adipose tissue is considered a possible cause of obesity. However, transgenic mice that overexpress the human LPL gene showed no increase in fat deposition as compared with controls. In the present study, we investigated effects of LPL on fat accumulation. Respiratory quotients and uptake of [3H] triolein by tissues (white and brown adipose tissue, and skeletal muscles) did not differ significantly for transgenic and non-transgenic mice. The mRNA levels of hormone-sensitive lipase (HSL) and HSL activity in adipose tissue during feeding were higher in LPL transgenic mice than in controls. Results suggest that the overexpression of LPL does not induce obesity by enhancing the hydrolysis of triglycerides in adipose tissue.

Adipose Tissue↗
PubMed1995

Increased expression of AML1 during retinoic-acid-induced differentiation of U937 cells.

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 the AML1 gene, three proteins, AML1a, AML1b and AML1c, are produced by alternative splicings. We previously showed that AML1 is potentially involved in myeloid cell differentiation. Here we analyzed the expression of AML1 in myeloid cell lines. It was revealed that AML1b and AML1c are the major AML1 proteins in these cell lines and that prior to morphological and functional differentiation, their expressions increase in U937 cells when treated with all-trans retinoic acid. These data suggest that the expression of AML1 is associated with myeloid cell differentiation.

Alternative Splicing↗
PubMed1995

No evidence for mutations in a putative subunit of the beta-cell ATP-sensitive potassium channel (K-ATP channel) in Japanese NIDDM patients.

The ATP-sensitive K channel (K-ATP channel) in pancreatic beta cells is believed to play a crucial role in glucose-stimulated insulin release. We investigated whether defects in the recently cloned gene for a putative subunit of this channel (KATP-2) could be a cause of diabetes in Japanese patients. The coding region of this beta-cell type channel gene was investigated in 192 diabetics with a family history of the disorder by single-stranded conformational polymorphism (SSCP) analysis. Two silent polymorphisms were found and confirmed by sequencing, but no missense or nonsense mutations were detected. The allele frequency of the polymorphisms was compared with 96 control subjects without a family history of the disease, and no clear difference was found. These results indicate that genetic defects of the KATP-2 channel may not be a major cause of diabetes in Japan.

Adult↗
PubMed1995

Integrin-mediated cell adhesion promotes tyrosine phosphorylation of p130Cas, a Src homology 3-containing molecule having multiple Src homology 2-binding motifs.

p130Cas (Cas) has been recently identified as a 130-kDa protein that is highly phosphorylated on tyrosine residues and is stably associated with p47v-crk (v-Crk) and p60v-src (v-Src) oncogene products in cells transformed by the respective genes. Cas is a novel signaling molecule having a single Src homology (SH) 3 domain and a cluster of multiple SH2-binding motifs. While the tight association of Cas with v-Crk and v-Src is strongly suggestive of a significant role in regulating cellular transformation, the function of Cas in normal untransformed cells is totally unknown. We report here that cell adhesion to fibronectin rapidly promotes tyrosine phosphorylation of Cas in human and rat fibroblast cell lines. The response was equally induced by cell adhesion to plates coated with vitronectin, laminin, and collagen but not by cell attachment to nonspecific substrate poly-L-lysine. The kinetic profile of Cas phosphorylation was almost identical with that of tyrosine phosphorylation of focal adhesion kinase pp125FAK (Fak), which is well known to be activated subsequent to integrin-mediated cell adhesion. Adhesion-dependent Cas phosphorylation was completely inhibited by treating cells with cytochalasin D, an agent that disrupts polymerization of actin stress fibers. These results suggest that tyrosine phosphorylation of Cas is stimulated by normal cell adhesion in close association with Fak phosphorylation and the formation of actin stress fibers. In v-Src- or v-Crk-transformed cells, however, the tyrosine phosphorylation of Cas is markedly increased in an adhesion-independent manner that is insensitive to treatment with cytochalasin D. Thus, Cas plays a role in signaling pathways mediated by cell adhesion as well as by transformation. We propose that Cas may amplify and propagate integrin-mediated signals by interacting with SH2-containing molecule(s).

Animals↗
PubMed1995

Expression of p210bcr/abl by metallothionein promoter induced T-cell leukemia in transgenic mice.

The p210bcr/abl chimeric protein is considered to be implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. To investigate its biologic function in vivo, we generated transgenic mice expressing p210bcr/abl driven by the metallothionein enhancer/promoter. Two of six founder mice and the transgenic progeny developed leukemias several months after birth. In the leukemic tissues, the expression of the p210bcr/abl transgene product was detected and the increased tyrosine-phosphorylation of cellular proteins was observed. The expressed p210bcr/abl transgene product was shown to possess an enhanced kinase activity. The leukemic cells showed rearrangements in the T-cell receptor loci, indicating that the leukemic cells were monoclonal and committed to the T-cell lineage. Polymerase chain reaction analysis for tissue distribution of p210bcr/abl expression showed that, in the transgenic line that reproducibly developed leukemias, p210bcr/abl was expressed in the hematopoietic tissues such as thymus and spleen; on the other hand, in the transgenic lines that have not developed leukemias, p210bcr/abl expression was detected only in the nonhematopoietic tissues such as the brain and kidney. These results suggest that the tumorigenicity of the p210bcr/abl chimeric protein is restricted to the hematopoietic tissues in vivo and that an event enhancing p210bcr/abl expression contributed a proliferative advantage to hematopoietic precursor cells and eventually developed T-cell leukemia in transgenic mice.

Animals↗
PubMed1995

The proto-oncogene product c-Cbl becomes tyrosine phosphorylated by stimulation with GM-CSF or Epo and constitutively binds to the SH3 domain of Grb2/Ash in human hematopoietic cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) are hematopoietic growth factors that regulate proliferation and differentiation of hematopoietic cells. They elicit and control a cascade of biochemical events, the earliest of which is tyrosine phosphorylation of several cellular proteins. Grb2/Ash is composed of SH2 and SH3 domains. The SH2 domain binds to tyrosine-phosphorylated proteins, and the SH3 domains bind to proteins containing proline-rich regions. It is considered that Grb2/Ash functions as an adapter protein linking tyrosine kinases and Ras in downstream of receptors for growth factors in fibroblasts. However, the mechanisms of signal transduction through Grb2/Ash and the roles of proteins associated with Grb2/Ash remain to be determined in hematopoietic cells. By means of the binding experiments using the glutathione S-transferase fusion protein including the full-length Grb2/Ash, we have found that Shc and unidentified 130- and 135-kDa proteins are associated with Grb2/Ash and that they are tyrosine phosphorylated by treatment with GM-CSF or Epo in a human leukemia cell line, UT-7. We have purified the 130-kDa protein (pp130) using the glutathione S-transferase-Grb2/Ash affinity column. The amino acid sequence analysis of the three peptides derived from the in situ protease digestion of the purified pp130 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, and the binding of Grb2/Ash to c-Cbl or Sos was not altered by GM-CSF stimulation. Moreover, c-Cbl (pp130) becomes tyrosine phosphorylated rapidly and transiently depending on GM-CSF or Epo stimulation. These findings strongly suggest that c-Cbl is implicated in the signal transduction of GM-CSF or Epo in hematopoietic cells and that c-Cbl is involved in another signaling pathway different from the Ras signaling pathway.

Adaptor Proteins, Signal Transducing↗