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Kyoko Toda

Publications and source records attributed to Kyoko Toda.

16 recordsLinked to original sources
PubMed2006

A single-base deletion in soybean flavonol synthase gene is associated with magenta flower color.

The Wm locus of soybean [Glycine max (L.) Merr.] controls flower color. Dominant Wm and recessive wm allele of the locus produce purple and magenta flower, respectively. A putative full-length cDNA of flavonol synthase (FLS), gmfls1 was isolated by 5' RACE and end-to-end PCR from a cultivar Harosoy with purple flower (WmWm). Sequence analysis revealed that gmfls1 consisted of 1,208 nucleotides encoding 334 amino acids. It had 59-72% homology with FLS proteins of other plant species. Conserved dioxygenase domains A and B were found in the deduced polypeptide. Sequence comparison between Harosoy and Harosoy-wm (magenta flower mutant of Harosoy; wmwm) revealed that they differed by a single G deletion in the coding region of Harosoy-wm. The deletion changed the subsequent reading frame resulting in a truncated polypeptide consisting of 37 amino acids that lacked the dioxygenase domains A and B. Extracts of E. coli cells expressing gmfls1 of Harosoy catalyzed the formation of quercetin from dihydroquercetin, whereas cell extracts expressing gmfls1 of Harosoy-wm had no FLS activity. Genomic Southern analysis suggested the existence of three to four copies of the FLS gene in the soybean genome. CAPS analysis was performed to detect the single-base deletion. Harosoy and Clark (WmWm) exhibited longer fragments, while Harosoy-wm had shorter fragments due to the single-base deletion. The CAPS marker co-segregated with genotypes at Wm locus in a F(2) population segregating for the locus. Linkage mapping using SSR markers revealed that the Wm and gmfls1 were mapped at similar position in the molecular linkage group F. The above results strongly suggest that gmfls1 represents the Wm gene and that the single-base deletion may be responsible for magenta flower color.

Amino Acid Sequence↗
PubMed2006

Cyclooxygenase-2 inhibitor and interferon-beta synergistically induce apoptosis in human hepatoma cells in vitro and in vivo.

Recent clinical trials have shown that interferon (IFN) is effective for chemoprevention against hepatocellular carcinoma (HCC). However, it remains controversial as to whether IFN exerts direct cytotoxicity against HCC. Cyclooxygenase (COX)-2 also plays a role in hepatocarcinogenesis and may mediate resistance to apoptosis in HCC. Therefore, we aimed to elucidate the combined effect of COX-2 inhibitor, NS-398, and IFN on in vitro growth suppression of HCC using 3 hepatoma cell lines (HepG2, PLC/PRF/5, and Huh7) and in vivo nude mouse xenotransplantation model using Huh7 cells. Only minimal growth inhibition was observed after treatment with IFN-beta alone in the 3 hepatoma cell lines. In contrast, treatment with NS-398 and IFN-beta synergistically inhibited cell proliferation in dose- and time-dependent manner. Apoptosis was identified by 4',6-diamidino-2-phenylindole dihydrochloride and fluorescent staining. IFN-beta up-regulated the expression of TRAIL, while NS-398 increased the expression of TRAIL receptors (especially of death receptor 5). Subsequently, activation of caspase-8 and caspase-3 was observed following the treatment with NS-398 and IFN-beta. Blockade of TRAIL with a specific antibody attenuated this apoptosis. Furthermore, we found that IFN-beta up-regulated COX-2 expression in Huh7 cells, and NS-398 might suppress the up-regulated COX-2 activity downstream of IFN signaling. In vivo experiment showed the combined regimen with NS-398 and IFN-beta reduced the growth of xenotransplated HCCs in nude mice. In conclusion, NS-398 is sufficient to overcome IFN resistance in hepatoma cells through the TRAIL/TRAIL receptor pathway, therefore, the combination would appear to be a new therapeutic regimen for HCC.

Animals↗
PubMed2005

Fine mapping of the FT1 locus for soybean flowering time using a residual heterozygous line derived from a recombinant inbred line.

Fine-mapping of loci related to complex quantitative traits is essential for map-based cloning. A residual heterozygous line (RHL) of soybean (Glycine max) derived from a recombinant inbred line (RIL) was used for fine-mapping FT1, which is a major quantitative trait locus (QTL) responsible for soybean flowering time. The residual heterozygous line RHL1-156 was selected from the RILs that were derived from two distantly related varieties, Misuzudaizu and Moshidou Gong 503. The genome of RHL1-156 contains a heterozygous segment (approximately 17 cM) surrounding the FT1 locus but is homozygous in other regions, including three other loci affecting flowering time. A large segregating population of 1,006 individuals derived by selfing of RHL1-156 included two homozygous genotypes for the nearest marker of FT1 whose flowering time differed by 26 days. No continuous range of phenotypes was observed, in contrast to the F2 population, suggesting that a single FT1 locus affected the flowering time in the RHL1-156 line. Linkage analysis revealed that the FT1 locus mapped as a single Mendelian factor between two tightly linked DNA markers, Satt365 and GM169, at distances of approximately 0.1 cM and 0.4 cM, respectively. Our results show that a RHL derived from RILs can be used to fine-map a QTL and that RHLs can be an efficient tool for a systematic fine-mapping of QTLs.

Breeding↗
PubMed2005

New approaches to mechanism analysis for drug discovery using DNA microarray data combined with KeyMolnet.

We have developed a comprehensive information platform, named KeyMolnet, for drug discovery and life science research in the post-genome era. Using KeyMolnet, we show new approaches to research into the biological mechanism in DNA microarray analysis. Thanks to the DNA microarray technology, it is now possible to obtain very large quantities of gene expression data at a time. However, it is still difficult to extract meaningful information from such large quantities of data and to analyze the relationship between gene expression data and biological function. We therefore developed an advanced tool that can generate molecular networks upon demand, and beyond signaling "cross-talks," can connect them to physiological phenomena and medical and drug information. Here we show the methods of mechanism analysis using the DNA microarray data and KeyMolnet, as well as the possible mechanism of inducing apoptosis in the human promyelocytic leukemia cell line, HL-60, treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), using the time series of gene expression data from DNA microarray experiments. KeyMolnet enables practical approaches to research into biological mechanisms, which in turn contribute to new discoveries in the medical, pharmaceutical and life sciences.

Apoptosis↗
PubMed2004

Improvement in reliability of probabilistic test of significant differences in GeneChip experiments.

A probabilistic test (FUMI theory) for GeneChip experiments has been proposed for selecting the genes which show significant differences in the gene expression levels between a single pair of treatment and control. This paper describes that the reliability of the judgment by the FUMI theory can be enhanced, when the selected genes are referred to biomolecular-functional networks of a commercial database. The genes judged as being differently expressed are grouped into a cluster in the biomolecular networks. It is also demonstrated that false positive genes have a trend in the networks to be isolated from each other, and also away from the clustered genes, since the false positive genes are randomly selected.

Algorithms↗
PubMed2003

Hypercapnic acidosis attenuates endotoxin-induced nuclear factor-[kappa]B activation.

Although permissive hypercapnia improves the prognosis of patients with acute respiratory distress syndrome, it has not been conclusively determined whether hypercapnic acidosis (HA) is harmful or beneficial to sustained inflammation of the lung. The present study was designed to explore the molecular mechanism of HA in modifying lipopolysaccharide (LPS)-associated signals in pulmonary endothelial cells. LPS elicited degradation of inhibitory protein kappaB (IkappaB)-alpha, but not IkappaB-beta, resulting in activation of nuclear factor (NF)-kappaB in human pulmonary artery endothelial cells. Exposure to HA significantly attenuated LPS-induced NF-kappaB activation through suppressing IkappaB-alpha degradation. Isocapnic acidosis and buffered hypercapnia showed qualitatively similar but quantitatively smaller effects. HA did not attenuate the LPS-enhanced activation of activator protein-1. Following the reduced NF-kappaB activation, HA suppressed the mRNA and protein levels of intercellular adhesion molecule-1 and interleukin-8, resulting in a decrease in both lactate dehydrogenase release into the medium and neutrophil adherence to LPS-activated human pulmonary artery endothelial cells. In contrast, HA did not inhibit LPS-enhanced neutrophil expression of integrin, Mac-1. Based on these findings, we concluded that hypercapnic acidosis would have anti-inflammatory effects essentially through a mechanism inhibiting NF-kappaB activation, leading to downregulation of intercellular adhesion molecule-1 and interleukin-8, which in turn inhibits neutrophil adherence to pulmonary endothelial cells.

Acidosis, Respiratory↗
PubMed2003

VAChT-Cre. Fast and VAChT-Cre.Slow: postnatal expression of Cre recombinase in somatomotor neurons with different onset.

The cholinergic gene locus (CGL) consists of the genes encoding the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To establish a cholinergic-specific Cre-expressing mouse, we constructed a transgene expression vector (VAChT-Cre) with 11.3 kb human CGL in which a Cre-IRES-EGFP unit was inserted in the VAChT open reading frame. The activity of Cre, whose expression was driven by the VAChT promoter, was examined by crossing a reporter mouse (CAG-CAT-Z) in which expression of LacZ is activated upon Cre-mediated recombination. Transgenic lines with the VAChT-Cre construct displayed the restricted Cre expression in a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus, but absent in visceromotor and other central and peripheral cholinergic neurons. Cre expression was first observed at postnatal day 7 and later detected in approximately 40-60% of somatomotor neurons. Based on the onset of Cre expression, we generated two mouse lines (two alleles; VAChT-Cre. Fast and VAChT-Cre.Slow) in which Cre expression reaches maximal levels fast and slow, respectively. The use of VAChT-Cre mice should allow us to deliver Cre to a subset of postnatal motor neurons, thereby bypassing lethality and facilitating analysis of gene function in adult motor neurons.

Animals↗
PubMed2003

Distribution and localization of caveolin-1 in sinusoidal cells in rat liver.

Caveolin, the principal structural protein in caveolae, is involved in signal transduction. The aim of the present study was to clarify the distribution and ultrastructural localization of caveolin-1 in hepatic sinusoidal endothelial cells (SECs) and hepatic stellate cell (HSCs) by confocal microscopy and the electron immunogold method. Liver tissue sections were prepared from male Wistar rats. SECs and HSCs were isolated from rat livers by collagenase infusion. For immunohistochemistry, liver sections were reacted with anticaveolin-1 antibody. The localization and distribution of caveolin-1 were identified by confocal immunofluorescence. The ultrastructural localization of caveolin-1 on SECs and HSCs was identified by electron microscopy using the immunogold method. Immunohistochemical studies using liver tissues localized caveolin-1 in sinusoidal lining cells, bile canaliculi, portal vein, and hepatic artery. By confocal microscopy, caveolin-1 was mainly demonstrated at the Golgi complex in SECs and HSCs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae (SEF) and vesicles in SECs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of caveolae and vesicles in HSCs. We concluded that caveolin-1 is localized from SEFs to the Golgi complex in SECs and from caveolae to the Golgi complex in HSCs.

Animals↗
PubMed2003

Differential induction of serum interleukin-6 and -12 by interferon-alpha and -beta administration in chronic hepatitis C patients.

Interferon (IFN) is widely used to manage chronic hepatitis C virus (HCV) infections. It contributes to the production of various cytokines, and alters cytokine interactions in vivo. Among them, interleukin (IL)-12, is thought to shift the immune system to Th-1 dominance and to, therefore, have a beneficial effect on eradication of HCV. On the other hand, IL-6 seems to be related to the acute inflammatory phenomenon, such as fever accompanied with IFN therapy. We studied the time courses of the serum IL-12 and IL-6 levels of patients with HCV infections treated with either IFN-alpha or IFN-beta. On the first day, both IFN-alpha and IFN-beta significantly induced serum IL-6, but when the administration was repeated, the serum IL-6 levels gradually decreased in both the IFN-alpha and the IFN-beta groups. The serum IL-6 levels correlated well with the daily peak body temperature of the patients, which also went down with repeated IFN administration. Serum IL-12 production was induced by IFN-alpha not by IFN-beta. The induced IL-12 decreased to baseline by repeating IFN-alpha administration. These results suggested that IFN-alpha and IFN-beta induce cytokine production in a different manner, and that their effect on cytokine interactions is short-term and reduced by repeated administration. Therefore, the induction of serum IL-12 production by IFN did not seem to have a critical effect during the early stage of IFN therapy.

Journal Article↗
PubMed2003

Test of significant differences with a priori probability in microarray experiments.

A traditional method for comparing two expression levels of genes in microarray experiments is the two-sample t-test. Because of the difficulty in using a large number of microarrays, an alternative method is required which can provide a reliable judgment of the comparison from a small number of replicates, even from a single pair of control and treatment. We present a method for detecting the changes in the gene expression levels under two different conditions in microarray experiments. Our method targets a single experiment for each condition, while retaining the statistical advantages of the t-test. The new proposals are: 1) standard deviation (SD) estimates of the expression levels which are an indicator for significant differences are given a priori as a function of the expression levels; 2) the limit of detection (LOD) for the expression levels is used to eliminate the majority of genes expressed at extremely low levels. The a priori SD estimates are obtained from six replicates under a fixed condition and are shown to be the approximate, but proper description of the expression uncertainty covering diverse conditions (e.g., different samples (human and rat) and different DNA chips). The LOD is defined as three times blank SD according to the IUPAC recommendation. A cell line (HL60) which will undergo macrophage differentiation on treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) is taken as an example. Our method is compared with the t-test for the data on duplicate TPA experiments and the former alone is evaluated with the data on a single TPA experiment. The errors from sample preparation and instrumental analysis are discussed.

Animals↗
PubMed2002

GGA proteins associate with Golgi membranes through interaction between their GGAH domains and ADP-ribosylation factors.

ADP-ribosylation factors (ARFs) are a family of small GTPases that are involved in various aspects of membrane trafficking events. These include ARF1-ARF6, which are divided into three classes on the basis of similarity in the primary structure: Class I, ARF1-ARF3; Class II, ARF4 and ARF5; and Class III, ARF6. Previous studies identified a novel family of potential ARF effectors, termed GGA1-GGA3, which interact specifically with GTP-bound ARF1 and ARF3 and are localized to the trans-Golgi network (TGN) or its related compartment(s) (GGA is an abbreviation for Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding protein). In the present study we have shown that ARF proteins belonging to the three classes, ARF1, ARF5 and ARF6, can interact with all GGA proteins in a yeast two-hybrid assay, in vitro and in vivo. Segmentation of GGA proteins and isolation of GGA mutants defective in ARF binding have revealed that a limited region within the GGA homology domain, which is conserved in the GGA family, is essential for ARF binding. Expression in cells of GTPase-restricted mutants of ARF1 and ARF5 blocks dissociation of GGA proteins from membranes induced by brefeldin A. However, neither of the ARF mutants recruits GGA mutants defective in ARF binding. On the basis of these observations, we conclude that at least ARF1 (Class I) and ARF5 (Class II) in their GTP-bound state cause recruitment of GGA proteins on to TGN membranes. In contrast, on the basis of similar experiments, ARF6 (Class III) may be involved in recruitment of GGA proteins to other compartments, possibly early endosomes.

ADP-Ribosylation Factors↗
PubMed2002

Inhibition of growth of human hepatoma cells by dual-function antisense IL-6 oligonucleotides.

Hepatocellular lineage cell lines (two hepatoma and Chang liver cell lines) were found to produce interleukin-6 (IL-6). As the human hepatoma cell line, HCC-M expresses mRNA for both IL-6 and IL-6 receptor, we examined the possibility that IL-6 acted as an autocrine growth factor for HCC-M cells using two IL-6 antisense oligonucleotides (AS-1 and AS-2 oligomers) which were synthesized from different regions of an IL-6 cDNA clone. Both IL-6 antisense oligonucleotides inhibited the growth of HCC-M cells within 48 h (% inhibition by AS-1 and AS-2 oligomers was 53 and 21%, respectively). Although inhibition of HCC-M cell growth induced by AS-2 oligomer was restored by addition of exogenous recombinant IL-6 (rIL-6), the inhibition of growth induced by AS-1 oligomer was not fully restored by exogenous rIL-6, implicating the involvement of a nonantisense mechanism associated with four contiguous guanosine residues in this sequence. The inhibitory effect of AS-2 oligomer was attenuated after 72 h (% inhibition was 8%), whereas the AS-1 oligomer-induced inhibition of growth was sustained beyond 72 h (% inhibition was 38--39%). Therefore, these dual-function oligonucleotides that act via both an antisense and nonantisense mechanism may be of potential therapeutic value against hepatoma.

Journal Article↗
PubMed2002

A single-base deletion in soybean flavonoid 3'-hydroxylase gene is associated with gray pubescence color.

The T locus of soybean (Glycine max (L.) Merr.) controls pubescence and seed coat color and is presumed to encode flavonoid 3'-hydroxylase (F3'H). The dominant T and the recessive t allele of the locus produce brown and gray pubescence, respectively. PCR primers were constructed based on the sequence of a soybean EST clone homologous to the F3'H gene. A putative full-length cDNA, sf3'h1 was isolated by 3' and 5' RACE. Sequence analysis revealed that sf3'h1 consists of 1690 nucleotides encoding 513 amino acids. It had 68% and 66% homology with corresponding F3'H protein sequences of petunia and Arabidopsis, respectively. A conserved amino acid sequence of F3'H proteins, GGEK, was found in the deduced polypeptide. Sequence analysis of the gene from a pair of near-isogenic lines for T, To7B (TT, brown) and To7G (tt, gray) revealed that they differed by a single C deletion in the coding region of To7G. The deletion changed the subsequent reading frame resulting in a truncated polypeptide lacking the GGEK consensus sequence and the heme-binding domain. Genomic Southern analysis probed by sf3'h1 revealed restriction fragment length polymorphisms between cultivars with different pubescence color. Further, sf3'h1 was mapped at the same position with T locus on LG3(c2). PCR-RFLP analysis was performed to detect the single-base deletion. To7B and three cultivars with brown pubescence exhibited shorter fragments, while To7G and three cultivars with gray pubescence had longer fragments due to the single-base deletion. The PCR-RFLP marker co-segregated with genotypes at the T locus in a F2 population segregating for the T locus. The above results strongly suggest that sJ3'h1 represents the T gene of soybean responsible for pubescence color and that the single-base deletion may be responsible for gray pubescence color.

Alleles↗