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K Nagata

Publications and source records attributed to K Nagata.

At least 19 recordsLinked to original sources
PubMed1995

Three-dimensional solution structure of bombyxin-II an insulin-like peptide of the silkmoth Bombyx mori: structural comparison with insulin and relaxin.

The three-dimensional solution structure of bombyxin-II, an insulin-like two-chain peptide produced by the brain of the silkworm Bombyx mori, has been determined by simulated annealing calculations based on 535 distance constraints and 24 torsion-angle constraints derived from NMR data and three distance constraints of the disulfide bonds. To our knowledge, this is the first three-dimensional structure determined for an invertebrate insulin-related peptide. The root-mean-square deviations between the best 10 structures and the mean structure are 0.58(+/- 0.15) A for the backbone heavy atoms (N, C alpha, C) and 1.03(+/- 0.18) A for all non-hydrogen atom if less well-defined N and C termini (A1, A20, B(-2) to B4 and B23 to B25) are excluded. The overall main-chain structure of bombyxin-II is similar to that of insulin. However, there are significant conformational and functional differences in their B-chain C-terminal parts. The B-chain C-terminal part of bombyxin-II adopts an extension of the B-chain central helix like that of relaxin and is not required for bombyxin activity, while the corresponding part of insulin adopts a sharp turn and a beta-strand and is essential for insulin activity. This structure demonstrates that bombyxin-II is more closely related to relaxin than to insulin, and suggests that insulin might have evolved the additional receptor-recognition site in the B-chain C-terminal beta-strand to distinguish itself from bombyxin and relaxin. The structure of bombyxin-II thus provides novel insights into the receptor recognition and divergent molecular evolution of insulin-superfamily peptides.

Amino Acid Sequence↗
PubMed1995

Identification of the receptor-recognition surface of bombyxin-II, an insulin-like peptide of the silkmoth Bombyx mori: critical importance of the B-chain central part.

Bombyxin-II, a brain-secretory peptide of the silkmoth Bombyx mori, shares 40% sequence identify and the characteristics core structure with human insulin. In spite of the structural similarity, no cross-activity is observed between them. To localize the active region of bombyxin-II, we have synthesized chimeric molecules of bombyxin-II and human insulin, and examined their bombyxin activity. Two chimeric molecules, which were sequentially identical except for the B-chain central part, showed significantly different potencies in bombyxin activity. Solution structure determination of these chimeric molecules revealed that their B-chain central parts took similar main-chain conformation, but formed dissimilar patches on their molecular surfaces. Therefore, the surface patch formed by the central part of the bombyxin-II B-chain is of critical importance for recognition of the bombyxin receptor. The above results, together with other data on the structure-activity relationships of bombyxin, indicate that the receptor-recognition surface of bombyxin-II includes the A-chain N and C, termini in addition to the B-chain central part. Though bombyxin-II, human insulin and human relaxin 2 use the common surface as their receptor-recognition sites, each of the surface patches is characterized by the variety of involved side-chains. Insulin and relaxin involve additional parts for receptor recognition, particularly the B-chain C-terminal part and the extended A-chain N-terminal helix, respectively. In conclusion, these ligands have evolved their own specific mechanisms for receptor recognition while retaining the major recognition surface.

Amino Acid Sequence↗
PubMed1995

Stimulation of sky receptor tyrosine kinase by the product of growth arrest-specific gene 6.

Sky (also called Rse, Brt, and Tyro3) is a member of a subfamily of related receptor tyrosine kinases, including Axl/Ufo/Ark and c-Eyk/Mer. We obtained evidence that Gas6 (the product of growth arrest-specific gene 6) is a ligand of the Sky receptor tyrosine kinase. Gas6, but not protein S (an anticoagulant protein structurally similar to Gas6), specifically bound to the soluble form of Sky (Sky-Fc), composed of the extracellular domain of Sky fused to the Fc domain of human immunoglobulin G1. The native and recombinant Gas6, but not protein S, stimulated tyrosine phosphorylation of Sky ectopically expressed in Chinese hamster ovary cells. Stimulation of Sky in response to Gas6 was inhibited by Sky-Fc. The half-maximal concentration of Gas6 that stimulated Sky was about 1 nM. Thus, Gas6 as a ligand for Sky specifically binds to and stimulates Sky receptor tyrosine kinase.

Animals↗
PubMed1995

Interaction between influenza virus proteins and pine cone antitumor substance that inhibits the virus multiplication.

Fractions obtained from pine cone extract (PCE) of Pinus parviflora Sieb. et Zucc. have been shown to suppress the growth of influenza virus. The inhibitory effects of one of the fractions, Fraction VII, on the formation of RNA-viral protein complex and the viral RNA synthesis were investigated. The formation of M1-RNA or NP-RNA complex was inhibited when M1 or NP was preincubated with the PCE fraction. The in vitro viral RNA synthesis was inhibited by the PCE fraction, while this inhibitory effect was titrated out by the increasing concentration of M1 protein. These results suggest that the major target of the PCE fraction was M1 protein.

Antineoplastic Agents↗
PubMed1995

Morphological changes of BM-N4 cells induced by bombyxin, an insulin-related peptide of Bombyx mori.

Bombyxin is a brain secretory peptide of the silkmoth Bombyx mori whose amino acid sequence shows considerable sequence homology with vertebrate insulin-family peptides. We found that a Bombyx cell line, named BM-N4, showed morphological changes when bombyxin was added to the culture medium at doses as low as 10(-10) M. Bombyxin-treated cells displayed a series of morphological modifications: 1 to 2 weeks after introduction of bombyxin, the cells increased in size, then they trended to aggregate, or took a spindle shape. These changes of the cells were bombyxin specific, not induced by other vertebrate insulin-family peptides. To prove that these modifications of the cells are mediated by receptors on the cell surface of the cells, we performed a receptor binding assay using 125I-labeled bombyxin. Scatchard analysis of the binding assay indicated that this cell line has a single class of receptors for bombyxin with a Kd = 2.36 +/- 0.56 nM and each cell has 15,800 +/- 1400 binding sites on the cell surface.

Animals↗
PubMed1995

Tamoxifen directly stimulates the mineralization of human osteoblast-like osteosarcoma cells through a pathway independent of estrogen response element.

Tamoxifen, an estrogen antagonist, stimulated the mineralization of osteoblasts in vitro in a dose-dependent manner in the presence of inorganic phosphate. The mechanism of mineralization by tamoxifen was different from that by 1 alpha, 25-dihydroxyvitamin D3. In addition, tamoxifen did not activate estrogen-response-element-mediated transcription in osteoblasts. These findings suggest that tamoxifen directly stimulates the mineralization of osteoblasts through a pathway independent of the conventional estrogen-response-element-mediated gene expression.

Alkaline Phosphatase↗
PubMed1995

Replication factor encoded by a putative oncogene, set, associated with myeloid leukemogenesis.

DNA replication of the adenovirus genome complexed with viral core proteins is dependent on the host factor designated template activating factor I (TAF-I) in addition to factors required for replication of the naked genome. Recently, we have purified TAF-I as 39- and 41-kDa polypeptides from HeLa cells. Here we describe the cloning of two human cDNAs encoding TAF-I. Nucleotide sequence analysis revealed that the 39-kDa polypeptide corresponds to the protein encoded by the set gene, which is the part of the putative oncogene associated with acute undifferentiated leukemia when translocated to the can gene. The 41-kDa protein contains the same amino acid sequence as the 39-kDa protein except that short N-terminal regions differ in both proteins. Recombinant proteins, which were purified from extracts of Escherichia coli, expressing the proteins from cloned cDNAs, possessed TAF-I activities in the in vitro replication assay. A particular feature of TAF-I proteins is the presence of a long acidic tail in the C-terminal region, which is thought to be an essential part of the SET-CAN fusion protein. Studies with mutant TAF-I proteins devoid of this acidic region indicated that the acidic region is essential for TAF-I activity.

Amino Acid Sequence↗
PubMed1995

Stimulation of DNA transcription by the replication factor from the adenovirus genome in a chromatin-like structure.

Adenovirus (Ad) genome DNA is complexed with viral core proteins in the virus particle and in host cells during the early stages of infection. This DNA protein complex, called Ad core, is thought to be the template for transcription and DNA replication in infected cells. The Ad core functioned as template for DNA replication in the cell-free system consisting of viral replication proteins, uninfected HeLa nuclear extracts, and a novel factor, template activating factor-I (TAF-I) that we have isolated from uninfected HeLa cytoplasmic fractions. The Ad core did not function as an efficient template in the cell-free transcription system with nuclear extracts of uninfected HeLa cells. The addition of TAF-I resulted in the stimulation of transcription from E1A and ML promoters on the Ad core. TAF-I was required, at least, for the formation of preinitiation complexes. These observations suggest that, in addition to factors essential for transcription on naked DNA template, the factor such as TAF-I needed for replication of the Ad core is also required for transcription from the Ad genome in a chromatin-like structure.

Adenoviridae↗
PubMed1995

Transcriptional elements directing a liver-specific expression of P450/6 beta A (CYP3A2) gene-encoding testosterone 6 beta-hydroxylase.

The P450/6 beta A (CYP3A2) gene encoding a testosterone 6 beta-hydroxylase is expressed predominantly in liver and induced by the treatment of rats with various compounds. To understand the mechanism of the basal transcriptional activation of the CYP3A2 gene, the cis-acting elements in the proximal promoter region (-165 to -73) of the CYP3A2 gene were identified in this study. Nuclear extract from rat livers interacted with three sites, 6 beta A-A (-106 to -87), 6 beta A-B (-140 to -119) and 6 beta A-C (-163 to -145). These sites were detectable by DNase I footprinting and gel mobility shift assays and found to share nucleotide sequence similarity with each other (T(A/C)(A/C)N(A/G)AAG(G/T)(C/T)CA). Direct repeats of AGTTCA (-134 to -120) and AG(G/C)TCA (-162 to -148) are also detected in 6 beta A-B and 6 beta A-C sites, respectively. To elucidate the relationship of these sites with basal transcriptional activation of the CYP3A2 gene, varying lengths of the proximal promoter region (-164 to +41) fused to a CAT reporter gene were transfected in human hepatoma (HepG2) and mouse adrenal tumor (Y-1) cells. The relative level of CAT activity in HepG2 cells was slightly increased by the deletion of the 5'-portion from -164 to -111 bp, but was reduced to 14% of the control (the construct including from -110 to +41) by the deletion from -110 to -81 including the 6 beta A-A site. On the other hand, these deletions have no clear effect on the level of the activity in Y-1 cells. Substitution mutations at two nucleotides in the 6 beta A-A site resulted in the reduction of CAT activity in HepG2 cells to 12% of the activity in the wild-type construct. The interaction of an oligonucleotide corresponding to the 6 beta A-A site (-106 to -87) with liver nuclear factors was completely inhibited by the addition of a typical oligonucleotide for hepatocyte nuclear factor-4 (HNF-4) binding site (F. M. Sladek, W. Zhong, E. Lai, and J. E. Darnell, Jr., 1990, Genes Dev. 4, 2353-2365) but not of oligonucleotides corresponding to 6 beta A-B or 6 beta A-C sites. These results suggest an essential role of the binding of HNF-4 and/or HNF-4-related nuclear factors to the 6 beta A-A site on the basal transcriptional activation of the CYP3A2 gene in liver cells.

Animals↗
PubMed1995

Quercetin suppresses heat shock response by down regulation of HSF1.

A bioflavonoid quercetin suppressed the stress response in heat-shocked cells and we have investigated the inhibitory mechanism in this report. After treatment of cells with nonlethal concentrations of quercetin, the binding of heat shock factor (HSF) to the heat shock element (HSE) was inhibited as detected by gel shift assay. We examined whether quercetin inhibits heat shock response by inhibiting HSF trimer-formation and found it was not the case. Instead, pretreatment of the cells with quercetin caused a decrease in the level of HSF1, especially of the constitutively phosphorylated form. This decrease was more prominent in heat-shocked cells than in control cells. These data suggest that (1) quercetin does not affect the trimer formation of HSF1, and (2) the decline of the HSF1-HSE complex might be linked to the decrease of HSF1 levels caused by quercetin.

Blotting, Western↗
PubMed1995

Age-related attenuation of HSP47 heat response in fibroblasts.

The collagen-binding heat shock protein of molecular weight 47,000 (HSP47), resident in the endoplasmic reticulum (ER), is assumed to play a specific role as a molecular chaperon in the processing of procollagen molecules. The present investigation of age-related alteration in the HSP47 heat response in cultured murine and human fibroblasts revealed expression in cells with a low population doubling level (PDL) derived from young mice and people more inducible by heat treatment than those from older mice and people. On the other hand, cells with a high PDL showed a very low heat response in terms of HSP47 expression regardless of the donor age. Northern blot analysis of HSP47 m-RNA indicated that the age related attenuation of HSP47 expression was regulated by transcriptional mechanisms. Furthermore, immunofluorescent analysis using a monoclonal antibody against the carboxylterminal propeptide of type I procollagen revealed far greater retention of procollagen molecules in the ER lumen of cells from old persons than in those from young persons. This was particularly prominent in heat-treated cells from old persons, indicating the possibility that the observed decrease in HSP47 heat response might cause blockage of procollagen transport to the Golgi and therefore secretion.

Aging↗
PubMed1995

Identification of heterotrimeric GTP-binding proteins in human megakaryoblastic leukemia cell line, MEG-01, and their alteration during cellular differentiation.

Various heterotrimeric GTP-binding proteins (G proteins) are possible to have important functions in hematopoietic cells. However, there has been no information regarding their expression in magakaryoblasts and/or megakaryocytes. In the present study, protein contents of seven G protein alpha subunits (Gs alpha, Gi2 alpha, Gi3 alpha, Gz alpha, G11 alpha, Gq alpha and G12 alpha) and beta subunit in a human megakaryoblastic leukemia cell line, MEG-01, were analyzed by immunoblotting. Immature MEG-01 cells expressed the alpha subunits of Gs, Gi2, Gi3, Gz, G11 and G12 at protein molecule level. During the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced differentiation process, the contents of Gi2 alpha and Gi3 alpha increased, whereas the protein levels of Gz alpha, Gs alpha, G11 alpha and G12 alpha were observed to hardly change. beta subunit was also observed to be present in immature MEG-01 cells and to increase continuously throughout the differentiation process. For the expression of Gi2 alpha and beta subunits, chronic TPA-treatment was required although Rac2, a low M(r) GTP-binding protein, was expressed abundantly by only 30 min-TPA-treatment followed by 3 day-culture.

Blood Platelets↗
PubMed1995

Two endogenous methyl-alpha-D-glucopyranoside uptake activities in Xenopus oocytes.

Endogenous methyl-alpha-D-glucopyranoside (AMG) uptake in Xenopus oocytes was measured with 14C-labeled AMG. Two AMG uptake activities, one Na(+)-dependent and the other Na(+)-independent, were observed in oocytes incubated with 2 mM AMG. However, only Na(+)-dependent uptake was observed at 0.1 mM AMG. Na(+)-dependent AMG uptake was attributed to the endogenous Na+/glucose co-transporter since it was inhibited by phlorizin. On the other hand, Na(+)-independent AMG uptake was inhibited by cytochalasin B or 2-deoxy-D-glucose. This suggests that AMG can be transported by the endogenous facilitated glucose transporter as well as by the endogenous co-transporter. In addition, a substrate range of the endogenous co-transporter was examined by competition experiments.

Animals↗
PubMed1995

Formation of 2-amino-3-methylimidazo[4,5-f]quinoline- and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline-sulfamates by cDNA-expressed mammalian phenol sulfotransferases.

In rat liver cytosol systems, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) were converted into their sulfamates in the presence of 3'-phosphoadenosine 5'-phosphosulfate at rates of 51.2 and 50.7 pmol/min/mg cytosol in the male, and 23.7 and 22.5 pmol/min/mg cytosol in the female, respectively. IQ-sulfamate formation was low (0.24 pmol/min/mg cytosol) in human liver cytosols, and MeIQx-sulfamate was not detected (< 0.1 pmol/min/mg cytosol). These results suggest only a minor contribution of IQ- and MeIQx-sulfamate formation to the detoxification of both heterocyclic amines in humans. Using sulfotransferase cDNA-expression systems, a rat ST1A1 arylsulfotransferase has been shown to catalyze the formation of the sulfamates, suggesting a role of the ST1A type of sulfotransferase in the N-sulfation of heterocyclic amines.

Animals↗
PubMed1995

Inhibitory action of lansoprazole and its analogs against Helicobacter pylori: inhibition of growth is not related to inhibition of urease.

The proton pump inhibitors omeprazole and lansoprazole and its acid-activated derivative AG-2000, which are potent and specific inhibitors of urease of Helicobacter pylori (K. Nagata, H. Satoh, T. Iwahi, T. Shimoyama, and T. Tamura, Antimicrob. Agents Chemother. 37:769-774, 1993), inhibited the growth of H. pylori. The growth was inhibited not only in urease-positive clinical isolates but also in their urease-negative derivatives which had no urease polypeptides. AG-1789, a derivative of lansoprazole with no inhibitory activity against H. pylori urease, also inhibited the growth of both strains even more strongly than the urease inhibitors lansoprazole and AG-2000. Furthermore, the antibacterial activity of omeprazole and lansoprazole was not affected by glutathione or dithiothreitol, which completely abolished the inhibitory activity of lansoprazole against H. pylori urease. These results indicated that the inhibitory action of these compounds against the growth of H. pylori was independent from the inhibitory action against urease.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1995

A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock.

Administration of monoclonal anti-CD3 antibody to mice treated with Propionibacterium acnes induced secretion of a high level of gamma interferon (IFN-gamma) into the circulation system, while it induced no significant release in untreated mice. In order to analyze this high-level induction of IFN-gamma in these bacterium-treated mice, we investigated the factors that might be involved. An activity that induces IFN-gamma in T cells was observed in the liver extracts of mice treated with P. acnes and subsequently challenged with lipopolysaccharide. Here, we purified an IFN-gamma-inducing factor from the liver extract to homogeneity and characterized it. Its molecular mass was 18 to 19 kDa, and its pI was 4.9. The amino acid sequence of the NH2-terminal portion was determined and shown to have no similarities to any protein in the EMBL, GenBank, and PIR data bases. The same molecule was also demonstrated in the serum factor that was previously reported to have an IFN-gamma-inducing activity and to have an apparent molecular mass of 75 kDa. Moreover, the activity of this serum factor was recovered in the fraction containing the 18- to 19-kDa protein under reducing conditions and was shown to have the same NH2-terminal amino acid sequence as that of the factor from the liver extract. In addition to the ability to induce IFN-gamma, this protein augmented T-cell proliferation and NK activity in the spleen cells. Thus, several of its biological activities were apparently similar to those of interleukin-12. These results indicated that this novel protein, which exhibited marked costimulatory activity on IFN-gamma production in vitro, was elevated vivo in response to P. acnes treatment. This factor, probably released from the producing cells by lipopolysaccharide stimuli, may be involved in the high-level induction of IFN-gamma in the P. acnes-treated mice.

Amino Acid Sequence↗
PubMed1995

Actinobacillus actinomycetemcomitans Y4 capsular-polysaccharide-like polysaccharide promotes osteoclast-like cell formation by interleukin-1 alpha production in mouse marrow cultures.

The mechanism of osteoclast-like cell formation induced by periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (serotype b) capsular-polysaccharide-like polysaccharide (capsular-like polysaccharide) was examined in a mouse bone marrow culture system. When mouse bone marrow cells were cultured with A. actinomycetemcomitans Y4 capsular-like polysaccharide for 9 days, many multinucleated cells were formed. The multinucleated cells showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb the calcified dentine. In this study, we examined the effects of antisera to interleukins on the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. Monospecific anti-mouse recombinant interleukin-1 alpha (rIL-1 alpha) serum completely inhibited the formation of osteoclast-like cells in the presence of A. actinomycetemcomitans Y4 capsular-like polysaccharide. However, anti-mouse rIL-1 beta and anti-mouse rIL-6 sera showed no effect on osteoclast-like cell formation. IL-1 receptor antagonist significantly inhibited the osteoclast-like cell formation mediated by A. actinomycetemcomitans Y4 capsular-like polysaccharide in mouse marrow cultures. The bioactive IL-1 was detected in the culture media of mouse bone marrow cells stimulated with A. actinomycetemcomitans Y4 capsular-like polysaccharide. These results indicate that IL-1 alpha is involved in the mechanism of the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. We sought to determine whether osteoclast-like cell formation induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide could be modulated by the protein kinase inhibitors H8 and HA1004. The formation of osteoclast-like cells was suppressed by H8 and HA1004. These findings suggest that the signals by protein kinases may regulate osteoclast-like cell formation induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. Furthermore, a correlation between IL-1 alpha and prostaglandin E2 in the osteoclast recruitment induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide is discussed.

Acid Phosphatase↗