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Naoyuki Taniguchi

Publications and source records attributed to Naoyuki Taniguchi.

At least 109 records · Page 6Linked to original sources
PubMed2002

The action of N-acetylglucosaminyltransferase-V is prevented by the bisecting GlcNAc residue at the catalytic step.

Using a purified protein and bisected acceptor oligosaccharides, we demonstrate that N-acetylglucosaminyltransferase (GnT)-V transfers a N-acetylglucosamine residue via a beta1,6-linkage to the bisected oligosaccharides. We also kinetically characterized the substrate specificity of GnT-V with respect to the bisected oligosaccharide. Although the K(m) values for the bisected acceptors were comparable to that for a non-bisected acceptor, the V(max) values for the bisected acceptors were much lower than that for the non-bisected acceptor. These findings suggest that the acceptor specificity of GnT-V is determined by the catalytic process rather than by its binding to the substrate. It was also found that the presence of the 2-N-acetyl group in the bisecting monosaccharide moiety plays a critical role in determining the catalytic efficiency of the enzyme.

Acetylglucosamine↗
PubMed2002

Uncoupling protein 2 plays an important role in nitric oxide production of lipopolysaccharide-stimulated macrophages.

The expression of uncoupling protein 2 (UCP2) was reduced in macrophages after stimulation with lipopolysaccharide (LPS). The physiological consequence and the regulatory mechanisms of the UCP2 down-regulation by LPS were investigated in a macrophage cell line, RAW264 cells. UCP2 overexpression in RAW264 cells transfected with eukaryotic expression vector containing ucp2 cDNA markedly reduced the production of intracellular reactive oxygen species. Furthermore, in the UCP2 transfectant, nitric oxide (NO) synthesis, inducible NO synthase (NOS II) protein, NOS II mRNA, and NOS II promoter activity were definitely decreased after LPS stimulation compared with those in parental RAW264 or RAW264 cells transfected with the vector alone. Reporter assays suggested that an enhancer element was located in the region of intron 2 of the UCP2 gene and that the UCP2 expression was down-regulated not by the 7.3-kb promoter region but by the 5' region of the UCP2 gene containing two introns. Deletion of intron 2 resulted in the low transcriptional activities and abolishment of the LPS-associated negative regulation. In addition, the mRNA expression of transfected UCP2 was suppressed in RAW264 cells transfected with expression vector containing UCP2 genomic DNA, but was markedly increased in cells transfected with the vector containing UCP2 intronless cDNA. These findings suggest that the LPS-stimulated signals suppress UCP2 expression by interrupting the function of intronic enhancer, leading to an up-regulation of intracellular reactive oxygen species, which activate the signal transduction cascade of NOS II expression, probably to ensure rapid and sufficient cellular responses to a microbial attack.

Animals↗
PubMed2002

Early-glycation of apolipoprotein E: effect on its binding to LDL receptor, scavenger receptor A and heparan sulfates.

Glycation is responsible for disruption of lipoprotein functions leading to the development of atherosclerosis in diabetes. The effects of apolipoprotein E (apoE) glycation were investigated with respect to its interaction with receptors. The interaction of apoE with the low density lipoprotein receptor (LDL-R) and scavenger receptor A (SR-A) was measured by competition experiments performed using, respectively, on a human fibroblast cell line 125I-LDL, and on a murine macrophage cell line (J774) 125I-acetylated LDL, and unlabeled apoE/phospholipid complexes. Glycated apoE binding to heparin and heparan sulfates (HS) was assessed by surface plasmon resonance (SPR) technology. Site-directed mutagenesis was then performed on Lys-75, the major glycation site of the protein. The prepared mutant protein proved to be useful as a tool to study the role of Lys-75 in apoE glycation. The findings showed that, although glycation has no effect on apoE binding either to the LDL-R or to SR-A, it impairs its binding to immobilized heparin and HS. The glycation of Lys-75 was found to be proceed rapidly and contributed significantly to total protein glycation. We propose that, in the case of diabetes, glycation may lead to the atherogenicity of apoE-containing lipoproteins disturbing their uptake via the HS proteoglycan pathway.

Apolipoproteins E↗
PubMed2002

Altholactone, a novel styryl-lactone induces apoptosis via oxidative stress in human HL-60 leukemia cells.

Plant styryl-lactone derivatives isolated from Goniothalamus sp. are potential compounds for cancer chemotherapy. In this study, we have examined the mechanisms of apoptosis induced by altholactone, a stryl-lactone isolated from the Malaysian plant G. malayanus on human HL-60 promyelocytic leukemia cells. Flow cytometric analysis of the externalization of phosphatidylserine (PS) using the annexin V/PI method on altholactone treated HL-60 cells showed a concentration-dependent increase of apoptosis from concentrations ranging from 10.8 (2.5 microg/ml) to 172.4 microM (40 microg/ml). Pre-treatment with the antioxidant N-acetylcysteine (1 mM) completely abrogated apoptosis induced by altholactone, suggesting for the involvement of oxidative stress. Further flow cytometric assessment of the level of intracellular peroxides using the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) confirmed that altholactone induced an increase in cellular oxidative stress in HL-60 cells which was suppressed by N-acetylcysteine. In summary, our results demonstrate for the first time that altholactone induced apoptosis in HL-60 cells occurs via oxidative stress.

Acetylcysteine↗
PubMed2002

Significance of the association between heparin-binding epidermal growth factor-like growth factor and CD9 in human gastric cancer.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family. Juxtacrine activity of proHB-EGF (the membrane-anchored form of HB-EGF) has been shown to be significantly potentiated when it is coexpressed with CD9 in vitro. The purpose of our study was to investigate the issue of whether proHB-EGF and CD9 are coexpressed in gastric cancer. HB-EGF gene expression and protein production in human gastric cancers was investigated, and EGF receptor and CD9 expressions were also evaluated. HB-EGF mRNA levels in gastric cancers were elevated, compared with normal gastric tissues, especially in the intestinal type. ProHB-EGF immunoreactivity was detected primarily in the cytoplasm and plasma membrane of gastric cancer cells. Of 66 patients, 40 (60.6%) exhibited proHB-EGF immunoreactivity and the level of its expression was significantly associated with tumor status (p < 0.01) and histological differentiation (p < 0.001). In addition, proHB-EGF mRNA was detected at high levels in the intestinal type by in situ hybridization. CD9 immunoreactivity was found to be preserved in 26 of 36 patients (72.2%) and CD9 protein expression was inversely associated with lymph node status (p < 0.05). A significant correlation between its expression and histological differentiation (p < 0.01) was found, and the association of CD9 with proHB-EGF was increased in the intestinal type, as evidenced by an immunoprecipitation method. These results indicate that the coexpression of proHB-EGF and CD9 may be involved in the tumorigenesis and/or proliferation of gastric cancers in a juxtacrine manner.

Adult↗
PubMed2002

Paranodal junction formation and spermatogenesis require sulfoglycolipids.

Mammalian sulfoglycolipids comprise two major members, sulfatide (HSO3-3-galactosylceramide) and seminolipid (HSO3-3-monogalactosylalkylacylglycerol). Sulfatide is a major lipid component of the myelin sheath and serves as the epitope for the well known oligodendrocyte-marker antibody O4. Seminolipid is synthesized in spermatocytes and maintained in the subsequent germ cell stages. Both sulfoglycolipids can be synthesized in vitro by using the isolated cerebroside sulfotransferase. To investigate the physiological role of sulfoglycolipids and to determine whether sulfatide and seminolipid are biosynthesized in vivo by a single sulfotransferase, Cst-null mice were generated by gene targeting. Cst(-/-) mice lacked sulfatide in brain and seminolipid in testis, proving that a single gene copy is responsible for their biosynthesis. Cst(-/-) mice were born healthy, but began to display hindlimb weakness by 6 weeks of age and subsequently showed a pronounced tremor and progressive ataxia. Although compact myelin was preserved, Cst(-/-) mice displayed abnormalities in paranodal junctions. On the other hand, Cst(-/-) males were sterile because of a block in spermatogenesis before the first meiotic division, whereas females were able to breed. These data show a critical role for sulfoglycolipids in myelin function and spermatogenesis.

Animals↗
PubMed2002

Apolipoprotein E activates Akt pathway in neuro-2a in an isoform-specific manner.

Apolipoprotein E (apoE) is a ligand for members of the low density lipoprotein (LDL) receptor family, receptors highly expressed in neurons. A study of one of the mechanisms by which apoE might affect neuronal cell metabolism is reported herein. ApoE can induce Akt/protein kinase B phosphorylation in Neuro-2a via two different pathways. Both pathways are mediated by phosphatidylinositol 3-kinase and cAMP-dependent protein kinase. The first pathway is stimulated by apoE3 and E4, but not by E2, after a 1-h incubation. The process requires the binding of apoE to the heparan sulfate proteoglycan/LDL receptor-related protein complex. The second pathway is activated after a 2-h incubation of the cells, in another isoform-dependent manner (E2 = E3 dbl greater-than sign E4) and is mediated by calcium. Our results suggest that apoE might affect cell metabolism and survival in neurons in an isoform-specific manner by inducing novel signaling pathways.

Animals↗
PubMed2002

A secreted type of beta 1,6-N-acetylglucosaminyltransferase V (GnT-V) induces tumor angiogenesis without mediation of glycosylation: a novel function of GnT-V distinct from the original glycosyltransferase activity.

Angiogenesis is the first regulatory step of tumor progression. Herein, we report on some findings that show that beta1,6-N-acetylglucosaminyltransferase V (GnT-V) functions as an inducer of angiogenesis that has a novel and completely different function from the original function of glycosyltransferase. A secreted type of GnT-V protein itself promoted angiogenesis in vitro and in vivo at physiological concentrations. The highly basic domain of GnT-V induced the release of fibroblast growth factor-2 from heparan sulfate proteoglycan on the cell surface and/or extracellular matrix, leading to angiogenesis. These findings provide some novel information on the relationship between GnT-V and tumor metastasis. The inhibition of GnT-V secretion or its expression represents a novel potential strategy for the inhibition of tumor angiogenesis.

Amino Acid Sequence↗
PubMed2002

Prometastatic effect of N-acetylglucosaminyltransferase V is due to modification and stabilization of active matriptase by adding beta 1-6 GlcNAc branching.

Oligosaccharide moieties of glycoproteins are structurally altered during development, carcinogenesis, and malignant transformations. It is well known that beta1-6 GlcNAc branching, a product of UDP-GlcNAc alpha-mannoside beta1-6-N-acetylglucosaminyltransferase (GnT-V), is associated with malignant transformation as the results of such alterations. However, the mechanism by which beta1-6 GlcNAc branching is linked to metastasis remains unclear, because the identification of specific glycoprotein(s) that are glycosylated by GnT-V and its biological function have not been examined. We herein report that matriptase, which activates both urokinase-type plasminogen activator and hepatocyte growth factor, is a target protein for GnT-V. The overexpression of GnT-V in gastric cancer cells leads to severe peritoneal dissemination in athymic mice, which can be attributed to the increased expression of matriptase. This increase was due to the acquired resistance of matriptase to degradation, since it is glycosylated by GnT-V and a corresponding increase in the active form. These results indicate that this process is a key element in malignant transformation, as the direct result of oligosaccharide modification.

Acetylglucosamine↗
PubMed2002

Sulfotransferases and sulfated oligosaccharides.

Structural diversity of the sugar chains attached to proteins and lipids that arises from the variety of combinations of different monosaccharides, different types of linkages, branch formation and secondary modifications, such as sulfation, possesses a large amount of biological information. A number of proteoglycans, glycoproteins, and glycolipids contain sulfated carbohydrates. Their sulfate groups provide a negative charge and play a role in a specific molecular recognition process. The sulfation of oligosaccharides is catalyzed by the Golgi-associated sulfotransferases. Recent success in molecular cloning of these sulfotransferases has brought a breakthrough in the understanding of biological function of sulfated oligosaccharides in a variety of contexts. Investigations on the relationship of sulfated oligosaccharides to human diseases including hereditary deficiency, cancer, inflammation, and infection may provide hints for curing disastrous diseases.

Animals↗
PubMed2002

Increased expression of manganese superoxide dismutase is associated with that of nitrotyrosine in myopathies with rimmed vacuoles.

Oxidative stress has been suggested as one of the pathogenetic mechanisms of inclusion body myositis (IBM). To study the role of antioxidant enzymes in myopathies with rimmed vacuoles, we examined expressions of copper, zinc superoxide dismutase (Cu, Zn-SOD) and manganese superoxide dismutase (Mn-SOD), and the relationship between SODs and other proteins localized in rimmed vacuoles in muscle biopsy specimens from three cases of sporadic IBM and two of distal myopathy with rimmed vacuoles (DMRV) as well as eight control cases of myopathies without rimmed vacuoles. Immunoblot analysis showed distinct protein bands of both SODs in IBM and DMRV using subtype-specific antibodies. Intensities of immunoreactive bands for Mn-SOD in IBM and DMRV were stronger than those in the control cases. Immunohistochemistry disclosed accumulation of both SODs in vacuolated muscle fibers in all cases of IBM and DMRV. Immunoreactivity for Mn-SOD was often colocalized with that of nitrotyrosine, cytochrome oxidase, tau, and lysosome-associated membrane proteins 2 (LAMP-2) in vacuolated fibers. Some of the Cu, Zn-SOD-positive vacuolated fibers were associated with ubiquitin. The two SODs may have different roles for cell protection, and the expression of Mn-SOD is associated with nitric oxide-induced oxidative damage in myopathies with rimmed vacuoles.

Adult↗
PubMed2002

NF-kappaB activation in non-parenchymal liver cells after partial hepatectomy in rats: possible involvement in expression of heparin-binding epidermal growth factor-like growth factor.

BACKGROUND/AIMS: Nuclear factor (NF) B activation plays a critical role in the initiation of liver growth after partial hepatectomy (PH). However, the issue of where specifically NF-B is activated is unclear. We previously reported that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a hepatotrophic factor. The aims of this study were to identify NF-B-activated cells and to clarify their involvement in HB-EGF expression after PH. METHODS: Using rats, a two-thirds PH was performed, after which NF-B-activated cells and HB-EGF-positive cells were identified by Southwestern histochemistry or immunohistochemistry. NF-B binding activity and HB-EGF gene expression were analyzed in rat hepatocytes and non-parenchymal cells (NPC) in primary culture. RESULTS: NF-B was activated in Kupffer cells and sinusoidal endothelial cells prior to activation in hepatocytes. HB-EGF immunoreactivity was detected after NF-B activation in the sinusoidal cells from those localized in the periportal zones of hepatic lobules. HB-EGF gene expression by tumor necrosis factor was accompanied by an increase in NF-B binding activity in NPC but not in hepatocytes in primary culture, which was abolished by pyrrolidine dithiocarbamate, an inhibitor of NF-B activation. CONCLUSIONS; NF-B was activated in NPC prior to activation in hepatocytes. NF-B activation may be involved in HB-EGF expression in NPC after PH.

Animals↗
PubMed2002

Novel substrates for nitric oxide synthases.

Enzymatic generation of nitric oxide (NO) by nitric oxide synthase (NOS) consists of two oxidation steps. The first step converts L-arginine to N(G)-hydroxy-L-arginine (NOHA), a key intermediate, and the second step converts NOHA to NO and L-citrulline. To fully probe the substrate specificity of the second enzymatic step, an extensive structural screening was carried out using a series of N-alkyl (and N-aryl) substituted-N'-hydroxyguanidines (1-14). Among the eleven N-alkyl-N'-hydroxyguanidines evaluated, N-n-propyl (2), N-iso-propyl (3), N-n-butyl (4), N-s-butyl (5), N-iso-butyl (6), N-pentyl (8) and N-iso-pentyl (9) derivatives were efficiently oxidized by the three isoenzymes of NOS (nNOS, iNOS and eNOS) to generate NO. N-Butyl-N'-hydroxyguanidine (4) was the best substrate for iNOS (K(m)=33 microM) and N-iso-propyl-N'-hydroxyguanidine (3) was the best substrate for nNOS (K(m)=56 microM). When the alkyl substituents were too small (such as ethyl 1) or too large (such as hexyl 10 and cyclohexyl 11), the activity decreased significantly. This suggests that the van der Waals interaction between the alkyl group and the hydrophobic cavity in the NOS active site contributes significantly to the relative reactivity of compounds 3-11. Moreover, five N-aryl-N'-hydroxyguanidines were found to be good substrates for iNOS, but not substrates for eNOS and nNOS. N-phenyl-N'-hydroxyguanidine was the best substrate among them (K(m)=243 microM). This work demonstrates that N-alkyl substituted hydroxyguanidine compounds are novel NOS substrates which 'short-circuit' the first oxidation step of NOS, and N-aryl substituted hydroxyguanidine compounds are isoform selective NOS substrate.

Animals↗
PubMed2002

A catalytically inactive beta 1,4-N-acetylglucosaminyltransferase III (GnT-III) behaves as a dominant negative GnT-III inhibitor.

beta 1,4-N-Acetylglucosaminyltransferase III (GnT-III) plays a regulatory role in the biosynthesis of N-glycans, and it has been suggested that its product, a bisecting GlcNAc, is involved in a variety of biological events as well as in regulating the biosynthesis of the oligosaccharides. In this study, it was found, on the basis of sequence homology, that GnT-III contains a small region that is significantly homologous to both snail beta 1,4GlcNAc transferase and beta1,4Gal transferase-1. Subsequent mutational analysis demonstrated an absolute requirement for two conserved Asp residues (Asp321 and Asp323), which are located in the most homologous region of rat GnT-III, for enzymatic activity. The overexpression of Asp323-substituted, catalytically inactive GnT-III in Huh6 cells led to the suppression of the activity of endogenous GnT-III, but no significant decrease in its expression, and led to a specific inhibition of the formation of bisected sugar chains, as shown by structural analysis of the total N-glycans from the cells. These findings indicate that the mutant serves a dominant negative effect on a specific step in N-glycan biosynthesis. This type of 'dominant negative glycosyltransferase', identified has potential value as a powerful tool for defining the precise biological roles of the bisecting GlcNAc structure.

Amino Acid Sequence↗
PubMed2002

Plasma 3-deoxyglucosone elevation in chronic renal failure is associated with increased aldose reductase in erythrocytes.

BACKGROUND: Serum concentrations of 3-deoxyglucosone (3DG), a highly reactive dicarbonyl compound, are elevated in uremic patients. Aldose reductase (AR) is an enzyme involved in both the detoxification of 3DG and producing precursors of 3DG. METHODS: We examined the relationship between plasma 3DG and erythrocyte AR content in uremic patients. Patients were divided into three groups: (1) progressive renal disease without hemodialysis (HD; chronic renal failure [CRF] group), (2) patients without diabetes mellitus (DM) treated with maintenance HD (HD group), and (3) patients with DM treated with maintenance HD (DM-HD group). High-performance liquid chromatography was used to measure 3DG, and erythrocyte AR was measured by means of enzyme-linked immunosorbent assay. RESULTS: Both 3DG and erythrocyte AR levels were significantly greater in the CRF, HD, and DM-HD groups than in healthy controls. These results did not change after HD sessions in the HD or DM-HD groups. Serum creatinine levels correlated with 3DG and erythrocyte AR levels in the control and CRF groups (3DG: r = 0.67; P < 0.001; erythrocyte AR: r = 0.71; P < 0.001). Both erythrocyte AR and 3DG levels then increased as renal function declined. A positive correlation was seen between 3DG and erythrocyte AR levels in all groups (r = 0.65; P < 0.001), and also between plasma osmolality and erythrocyte AR level (r = 0.46; P < 0.001). CONCLUSION: Both erythrocyte AR and 3DG levels are increased in uremic patients, and these increases could possibly contribute to the development of uremic symptoms.

Adult↗
PubMed2002

A glycomic approach to hepatic tumors in N-acetylglucosaminyltransferase III (GnT-III) transgenic mice induced by diethylnitrosamine (DEN): identification of haptoglobin as a target molecule of GnT-III.

A glycomic approach to the identification of target molecules in glycosyltransferase gene targeting mice is a promising strategy to understand the biological significance of glycosyltransferase genes in vivo. In order to understand the biological effects of N-acetylglucosaminyltransferase III (GnT-III) on tumor formation in the liver, diethylnitrosamine (DEN) induced tumor formation in the GnT-III transgenic mice was examined. Our findings show that the incidence of hepatic tumor could be dramatically suppressed. A glycomic approach using two-dimensional gel electrophoresis followed by lectin blot analysis and sequence analysis revealed that haptoglobin, a radical scavenger molecule in serum was heavily glycosylated in hepatic tumor-bearing GnT-III transgenic mice that had been treated with DEN. Immunoprecipitation followed by E4-PHA lectin blot analysis also confirmed that the bisecting GlcNAc, a product of GnT-III was added to haptoglobin molecules. Since the use of DEN is known to lead to the production of lipid peroxidation products which facilitate this reaction and haptoglobin is an acute phase reactant, acting as a radical scavenger against hemoglobin or iron stimulated lipid peroxidation, a relationship between the glycosylation of haptoglobin and the suppression of hepatoma development can not be ruled out. This paper is the first report that shows a relationship between the sugar chains of glycoproteins with radical scavenger activity and hepatocarcinogenesis.

Animals↗
PubMed2002

Differential expression of glutathione reductase and cytosolic glutathione peroxidase, GPX1, in developing rat lungs and kidneys.

A mutant rat GPX1 (a cytosolic predominant form), in which the selenocysteine residue in the catalytic center was replaced by cysteine, was prepared and an antibody against the mutant enzyme was raised. The resultant antibody specifically reacted with rat GPX1 and was, together with the Glutathione reductase (GR) antibody, used in a Western blot analysis and immunohistochemistry experiments. To elucidate the physiological coupling of these enzymes under oxidative stress which accompanies the birth, developmental changes of the protein levels and enzymatic activities of GR and GPX1 were examined for lungs and kidneys from prenatal fetus to adult rats. The expression of GR was already evident at the prenatal stage and remained high in lungs at all stages. However, GR activity in kidneys gradually increased after birth reaching maximal levels at adulthood. An immunohistochemical study showed that GR was strongly bound to the bronchial epithelia in lungs and the epithelial cells of renal tubes. GPX1 was expressed in the renal tube epithelial cells and its level gradually increased after birth in a manner similar to that of GR. The expression of GPX1 in the lungs was, on the other hand, variable and occurred in some alveolar cells and bronchial epithelia only at restricted periods. It preferentially localized in nuclei at a late stage of development. Thus, the expression of the two functionally coupled enzymes via GSH did not appear to coordinate with development, tissue localization or under oxidative stress. Since many gene products show GSH-dependent preoxidase activity, other peroxidase(s) may be induced to compensate for the low GPX1 levels at stages with high GR expression.

Animals↗