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Biomedical subjects

Hideki Takahashi

Publications and source records attributed to Hideki Takahashi.

At least 19 recordsLinked to original sources

Arabidopsis SLIM1 is a central transcriptional regulator of plant sulfur response and metabolism.

Sulfur is an essential macronutrient required for plant growth. To identify key transcription factors regulating the sulfur assimilatory pathway, we screened Arabidopsis thaliana mutants using a fluorescent reporter gene construct consisting of the sulfur limitation-responsive promoter of the SULTR1;2 sulfate transporter and green fluorescent protein as a background indicator for monitoring plant sulfur responses. The isolated mutant, sulfur limitation1 (slim1), was unable to induce SULTR1;2 transcripts under low-sulfur (-S) conditions. Mutations causing the sulfur limitation responseless phenotypes of slim1 were identified in an EIL family transcription factor, ETHYLENE-INSENSITIVE3-LIKE3 (EIL3), whose functional identity with SLIM1 was confirmed by genetic complementation. Sulfate uptake and plant growth on -S were significantly reduced by slim1 mutations but recovered by overexpression of SLIM1. SLIM1 functioned as a central transcriptional regulator, which controlled both the activation of sulfate acquisition and degradation of glucosinolates under -S conditions. Metabolite analysis indicated stable accumulation of glucosinolates in slim1 mutants, even under -S conditions, particularly in the molecular species with methylsulfinylalkyl side chains beneficial to human health. Overexpression of SLIM1 and its rice (Oryza sativa) homologs, but no other EIL genes of Arabidopsis, restored the sulfur limitation responseless phenotypes of slim1 mutants, suggesting uniqueness of the SLIM1/EIL3 subgroup members as sulfur response regulators.

Amino Acid Sequence↗

Additive contribution of AMT1;1 and AMT1;3 to high-affinity ammonium uptake across the plasma membrane of nitrogen-deficient Arabidopsis roots.

In Arabidopsis four root-expressed AMT genes encode functional ammonium transporters, which raises the question of their role in primary ammonium uptake. After pre-culturing under nitrogen-deficiency conditions, we quantified the influx of (15)N-labeled ammonium in T-DNA insertion lines and observed that the loss of either AMT1;1 or AMT1;3 led to a decrease in the high-affinity ammonium influx of approximately 30%. Under nitrogen-sufficient conditions the ammonium influx was lower in Columbia glabra compared with Wassilewskija (WS), and AMT1;1 did not contribute significantly to the ammonium influx in Col-gl. Ectopic expression of AMT1;3 under the control of a 35S promoter in either of the insertion lines amt1;3-1 or amt1;1-1 increased the ammonium influx above the level of their corresponding wild types. In transgenic lines carrying AMT-promoter-GFP constructs, the promoter activities of AMT1;1 and AMT1;3 were both upregulated under nitrogen-deficiency conditions and were localized to the rhizodermis, including root hairs. AMT gene-GFP fusions that were stably expressed under the control of their own promoters were localized to the plasma membrane. The double insertion line amt1;1-1amt1;3-1 showed a decreased sensitivity to the toxic ammonium analog methylammonium and a decrease in the ammonium influx of up to 70% relative to wild-type plants. These results suggest an additive contribution of AMT1;1 and AMT1;3 to the overall ammonium uptake capacity in Arabidopsis roots under nitrogen-deficiency conditions.

Arabidopsis↗

Pollen of Ceratostema (Ericaceae, Vaccinieae): tetrads without septa.

The pollen morphology of two species of the Neotropical genus Ceratostema (Ericaceae) was examined by light, scanning and transmission electron microscopy. The Ceratostema species examined have 3-colporate pollen grains united in permanent tetrahedral tetrads that show a common condition encountered in the Ericaceae. But the septal exine was absent between two neighboring grains in each pollen tetrad of Ceratostema. The pollen tetrads without septa are the first report for the Ericaceae as well as other angiosperm families.

Ericaceae↗

Single amino acid alterations in Arabidopsis thaliana RCY1 compromise resistance to Cucumber mosaic virus, but differentially suppress hypersensitive response-like cell death.

Resistance to an yellow strain of Cucumber mosaic virus [CMV(Y)] in Arabidopsis thaliana ecotype C24 is conferred by the CC-NBS-LRR type R gene, RCY1. RCY1-conferred resistance is accompanied by a hypersensitive response (HR), which is characterized by the development of necrotic local lesion (NLL) at the site of infection that restricts viral spread. To further characterize the role of RCY1 in NLL formation we have identified six recessive CMV(Y)-susceptible rcy1 mutants, four of which contain single amino acid substitutions in RCY1: rcy1-2 contains a D to N substitution in the CC domain, rcy1-3 and rcy1-4 contain R to K and E to K substitutions, respectively, in the LRR domain, and rcy1-6 contains a W to C substitution in the NBS domain. The rcy1-5 and rcy1-7 contain nonsense mutations in the LRR and NBS domains, respectively. Although the virus systemically spread in all six rcy1 mutants, HR-associated cell death was differentially induced in these mutants. In comparison to the wild type C24 plant, HR was not observed in the CMV(Y)-inoculated leaves of the rcy1-3, rcy1-5, rcy1-6 and rcy1-7 mutants. In contrast, delayed NLL development was observed in the virus inoculated leaves of the rcy1-2 and rcy1-4 mutants. In addition, necrosis accompanied by elevated accumulation of PR gene transcript also appeared in the non-inoculated leaves of the rcy1-2 and rcy1-4 mutants. Trans-complementation was observed between the rcy1-2 and rcy1-4 alleles; in F1 plants derived from a cross between rcy1-2 and rcy1-4, HR associated cell death was accelerated and systemic spread of the virus was partially suppressed than in the homozygous rcy1-2 and rcy1-4 plants. Our results suggest that the CC, NBS and LRR domains of RCY1 are required for restriction of virus spread but differentially impact the induction of HR-like cell death. Furthermore, these results also predict inter-molecular interaction involving RCY1 in Arabidopsis resistance to CMV(Y).

Alleles↗

Identification of a novel Cys2/His2-type zinc-finger protein as a component of a spermine-signaling pathway in tobacco.

In a previous work, we identified a Cys(2)/His(2)-type zinc-finger transcription repressor, (ZFT1), that functions in a spermine-mediated signal transduction pathway in tobacco plants. Database search disclosed the presence of another Cys(2)/His(2)-type zinc-finger protein ZFP1 (accession number AAC06243) in tobacco plants. In this work, we characterized ZFP1 and investigated whether this protein is also involved in a Spm-signaling pathway. This factor showed the highest identity to petunia ZPT2-2 and higher similarity to petunia ZPT2-3, Arabidopsis STZ/ZAT10, soybean SCOF-1, red pepper CAZFP1/CaPIF1 as well as to tobacco ZFT1. ZFP1 localized to the nucleus and had a specific DNA-binding activity, supportive to be a transcription factor. Furthermore, the protein had a mild repression activity on transcription in plant cells. The expression of ZFP1, encoding ZFP1, was upregulated during tobacco mosaic virus-induced hypersensitive response. ZFP1 expression was also induced by exogenously applied spermine and its induction was repressed by inhibitors of amine oxidase/polyamine oxidase. Collectively, our data indicate that ZFP1 is a new transcription factor which functions in a spermine-signaling pathway in tobacco.

Amino Acid Sequence↗

Novel Rh catalysis in cross-coupling between alkyl halides and arylzinc compounds possessing ortho-COX (X = OR, NMe2, or Ph) groups.

[reaction: see text] Rh-dppf was found to be an efficient catalyst for the cross-coupling between primary alkyl halides bearing beta-hydrogens and arylzinc compounds possessing carbonyl groups such as ester, amide, or ketone at the ortho position. Various functional groups such as ester, nitrile, or acyloxylate moieties on the halides were tolerated under the catalysis conditions. Arylzinc compounds free of ortho-carbonyl groups reacted well with ethyl 3-iodopropanoate, suggesting that the essential intramolecular interaction between carbonyl groups and Rh promotes the reductive elimination.

Journal Article↗

Synthesis of functionalized benzylsilanes from arylzinc compounds and (iodomethyl)trimethylsilane by means of a novel Rh catalysis.

[reaction: see text] The catalytic activity of a Rh complex in cross-coupling between ArZnI and TMSCH2I was examined in which the Rh complex, generated in situ from [RhCl(1,5-cyclooctadiene)]2 and 1,1'-bis(diphenylphosphino)ferrocene, exhibited excellent catalytic activity for the production of various functionalized benzylsilanes in good yields. From 31P NMR studies of various solutions containing several of the reaction components, confirmation of the rapid and quantitative transfer of aryl groups from ArZnI to the Rh complex to form arylrhodium species was ascertained. A catalytic cycle, commencing with the transmetalation, was thus proposed for the reaction.

Journal Article↗

Downregulation of type II bone morphogenetic protein receptor in hypoxic pulmonary hypertension.

Heterozygous mutations in the type II receptor for bone morphogenetic protein (BMPR-II) and dysfunction of BMPR-II have been implicated in patients with primary pulmonary hypertension (PH). To clarify the possible involvement of BMP and BMPR-II in the development of hypoxic PH, the expression of BMP-2, BMPR-II, and their downstream signals were investigated in rat lung under normal and hypoxic conditions by RT-PCR, immunoblot, and immunohistochemical methods. In rats under normal conditions, BMP-2 is localized in the endothelium of the pulmonary artery, whereas BMPR-II is abundantly expressed in the endothelium, smooth muscle cells, and adventitial fibroblasts. After 0.5 and 3 days of exposure to hypoxia, upregulation of BMP-2 was observed in the intrapulmonary arteries. The change was accompanied by activation of its downstream signaling, p38 MAPK, and Erk1/2 MAPK, and the apoptotic process, measured by caspase-3 activity and TdT-mediated dUTP nick end labeling-positive cells. In contrast, a significant decrease in the expression of BMPR-II and inactivation of p38 MAPK and caspase-3 were observed in the pulmonary vasculature after 7-21 days of hypoxia exposure. Because BMP-2 is known to inhibit proliferation of vascular smooth muscle cells and promote cellular apoptosis, disruption of BMP signaling pathway through downregulation of BMPR-II in chronic hypoxia may result in pulmonary vascular remodeling due to the failure of critical antiproliferative/differentiation programs in the pulmonary vasculature. These results suggest abrogation of BMP signaling may be a common molecular pathogenesis in the development of PH with various pathophysiological events, including primary and hypoxic PH.

Animals↗

Gln49 and Ser174 residues play critical roles in determining the catalytic efficiencies of plant glutamine synthetase.

Two essential residues playing critical roles in determining the substrate specificities of cytosolic glutamine synthetase (GS1) have been identified from the alignment of high-affinity (GLN1;1 and GLN1;4) and low-affinity (GLN1;2 and GLN1;3) GS1 isoenzymes in Arabidopsis, and confirmed by site-directed mutagenesis. The results indicated that either K49Q or A174S mutation is sufficient to increase the catalytic efficiencies of GLN1;3 by decreasing its Km values for ammonium. In contrast, replacement of Gln49 and Ser174 by lysine and alanine, respectively, was detrimental to glutamine synthetic activities in GLN1;4. The results suggested that Gln49 and Ser174 in the high-affinity GS1 isoenzymes are interchangeable with Lys49 and Ala174 in the low-affinity variants at the corresponding positions.

Alanine↗

Overexpression of AtCpNifS enhances selenium tolerance and accumulation in Arabidopsis.

Selenium (Se) is an essential element for many organisms but is toxic at higher levels. CpNifS is a chloroplastic NifS-like protein in Arabidopsis (Arabidopsis thaliana) that can catalyze the conversion of cysteine into alanine and elemental sulfur (S0) and of selenocysteine into alanine and elemental Se (Se0). We overexpressed CpNifS to investigate the effects on Se metabolism in plants. CpNifS overexpression significantly enhanced selenate tolerance (1.9-fold) and Se accumulation (2.2-fold). CpNifS overexpressors showed significantly reduced Se incorporation into protein, which may explain their higher Se tolerance. Also, sulfur accumulation was enhanced by approximately 30% in CpNifS overexpressors, both on media with and without selenate. Root transcriptome changes in response to selenate mimicked the effects observed under sulfur starvation. There were only a few transcriptome differences between CpNifS-overexpressing plants and wild type, besides the 25- to 40-fold increase in CpNifS levels. Judged from x-ray analysis of near edge spectrum, both CpNifS overexpressors and wild type accumulated mostly selenate (Se(VI)). In conclusion, overexpression of this plant NifS-like protein had a pronounced effect on plant Se metabolism. The observed enhanced Se accumulation and tolerance of CpNifS overexpressors show promise for use in phytoremediation.

Arabidopsis↗

Genistein, a phytoestrogen, attenuates monocrotaline-induced pulmonary hypertension.

BACKGROUND: Pulmonary hypertension is characterized by high pulmonary blood pressure, vascular remodeling, and right ventricular hypertrophy. Although recent studies suggest that an imbalance between endothelial mediators on pulmonary vasculature may contribute to the development of pulmonary hypertension, the pathogenesis is not fully understood and the treatment of pulmonary hypertension is still unresolved. OBJECTIVE: The purpose of this study was to investigate whether genistein, a phytoestrogen derived from soybean, would prevent the development of monocrotaline (MCT)-induced pulmonary hypertension in rats. Hemodynamic parameters of catheterized rats and morphological feature of lungs were evaluated among MCT-treated rats receiving or not receiving genistein. Furthermore, examination of expression in endothelial nitric oxide synthase and endothelin-1 peptide level was performed. METHODS: Daily supplementation with either genistein (0.2 mg/kg) or vehicle was started 2 days prior to a single-dose injection of MCT (60 mg/kg). On day 28, rats underwent catheterization, and right ventricular hypertrophy and morphological features were assessed. Furthermore, endothelial nitric oxide synthase and endothelin-1 were examined by Western blot analysis and radioimmunoassay, respectively, in homogenated lungs. RESULTS: In rats that received daily supplementation of genistein, mean pulmonary arterial pressure was significantly reduced, whereas mean systemic arterial pressure and heart rate were unaltered compared with MCT control rats on day 28 after MCT injection. Right ventricular hypertrophy, medial wall thickness of pulmonary arteries corresponding to the terminal bronchioles, and the degree of neo-muscularization of more distal arteries were less severe in genistein-treated rats. Genistein supplementation improved MCT-induced downregulation of expression of endothelial nitric oxide synthase in the lungs. However, endothelin-1 peptide levels did not differ among all groups of lungs. CONCLUSIONS: We conclude that daily supplementation of genistein potently attenuates MCT-induced pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular remodeling in rats. The underlying mechanism responsible for this effect may be partly related to the restoration of a decreased expression of endothelial nitric oxide synthase.

Animals↗

Clinical features of third open-heart valve surgery at the same valve position.

OBJECTIVE: Recently, there has been an increase in case of repeated open-heart valve surgery and the clinical results of the second surgery are only slightly worse than those of the first surgery. However, clinical results of the third open-heart valve surgery at the same position are rarely reported. Clinical features of third open-heart valve surgery at the same position are discussed in this study. METHODS: Between 1995 and 2004, 16 patients underwent third open-heart valve surgery at the same valve position under cardiopulmonary bypass. The average age of the 16 patients, 12 females and 4 males, was 56 +/- 15 years. Clinical features of the 16 cases were retrospectively analyzed. RESULTS: Mechanical valve nonstructural dysfunction was the most common valve malady, followed by bioprosthetic valve dysfunction. The duration of surgery from skin incision to establishment of the cardiopulmonary bypass was 94 +/- 42 minutes. Myocardial ischemia time was 137 +/- 38 minutes and extracorporeal circulation time was 212 +/- 82 minutes. Early mortality was seen in 1 patient (6.25%) and late mortality was seen in 1 patient. CONCLUSION: Mechanical valve nonstructural valve dysfunction leads to repeated valve surgery. The clinical results of the third open-heart valve surgery at the same valve position are acceptable, and the mid-term survival is excellent.

Adult↗

Tobacco ZFT1, a transcriptional repressor with a Cys2/His2 type zinc finger motif that functions in spermine-signaling pathway.

We previously proposed that a spermine (Spm)-mediated signal transduction pathway is involved in the hypersensitive response induced by Tobacco mosaic virus (TMV) in tobacco plants. To identify regulatory component(s) of this pathway, we surveyed a tobacco cDNA library and found that the ZFT1 gene, which encodes a Cys2/His2 type zinc-finger protein, is Spm-responsive. ZFT1 was not induced by two other polyamines, putrescine and spermidine, or by salicylic acid (SA), jasmonic acid or ethylene. Furthermore, ZFT1 was upregulated in TMV- inoculated tobacco plants in an N gene-dependent manner. Notably, induction of ZFT1 by Spm and by TMV infection was unimpaired in NahG-transgenic tobacco plants, indicating that cross-talk with an SA signaling pathway is not involved in this response. Within the Spm-signaling pathway, we found that ZFT1 functioned downstream of both mitochondrial dysfunction and mitogen-activated protein kinase activation. The ZFT1 protein has two zinc finger motifs and shows a high degree of similarity to ZPT2-3 in petunia and SCOF1 in soybean. However, unlike the latter two proteins, ZFT1 binds to the EP1S sequence and functions as a transcription repressor. Moreover, interestingly, ZFT1 overexpression rendered tobacco plants more tolerant to TMV. Based on the results presented here, we propose that ZFT1 functions as a transcription repressor in a Spm signaling pathway, thereby accelerating necrotic local region formation in tobacco leaves.

Amino Acid Sequence↗

Identification of a novel cis-acting element conferring sulfur deficiency response in Arabidopsis roots.

SULTR1;1 high-affinity sulfate transporter is highly regulated in the epidermis and cortex of Arabidopsis roots responding to sulfur deficiency (-S). We identified a novel cis-acting element involved in the -S-inducible expression of sulfur-responsive genes in Arabidopsis. The promoter region of SULTR1;1 was dissected for deletion and gain-of-function analysis using luciferase (LUC) reporter gene in transgenic Arabidopsis. The 16-bp sulfur-responsive element (SURE) from -2777 to -2762 of SULTR1;1 promoter was sufficient and necessary for the -S-responsive expression, which was reversed when supplied with cysteine and glutathione (GSH). The SURE sequence contained an auxin response factor (ARF) binding sequence (GAGACA). However, SURE was not responsive to naphthalene acetic acid, indicating its specific function in the sulfur response. The base substitution analysis indicated the significance of a 5-bp sequence (GAGAC) within the conserved ARF binding site as a core element for the -S response. Microarray analysis of early -S response in Arabidopsis roots indicated the presence of SURE core sequences in the promoter regions of -S-inducible genes on a full genome GeneChip array. It is suggested that SURE core sequences may commonly regulate the expression of a gene set required for adaptation to the -S environment.

Anion Transport Proteins↗

Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor.

The integration of metabolomics and transcriptomics can provide precise information on gene-to-metabolite networks for identifying the function of unknown genes unless there has been a post-transcriptional modification. Here, we report a comprehensive analysis of the metabolome and transcriptome of Arabidopsis thaliana over-expressing the PAP1 gene encoding an MYB transcription factor, for the identification of novel gene functions involved in flavonoid biosynthesis. For metabolome analysis, we performed flavonoid-targeted analysis by high-performance liquid chromatography-mass spectrometry and non-targeted analysis by Fourier-transform ion-cyclotron mass spectrometry with an ultrahigh-resolution capacity. This combined analysis revealed the specific accumulation of cyanidin and quercetin derivatives, and identified eight novel anthocyanins from an array of putative 1800 metabolites in PAP1 over-expressing plants. The transcriptome analysis of 22,810 genes on a DNA microarray revealed the induction of 38 genes by ectopic PAP1 over-expression. In addition to well-known genes involved in anthocyanin production, several genes with unidentified functions or annotated with putative functions, encoding putative glycosyltransferase, acyltransferase, glutathione S-transferase, sugar transporters and transcription factors, were induced by PAP1. Two putative glycosyltransferase genes (At5g17050 and At4g14090) induced by PAP1 expression were confirmed to encode flavonoid 3-O-glucosyltransferase and anthocyanin 5-O-glucosyltransferase, respectively, from the enzymatic activity of their recombinant proteins in vitro and results of the analysis of anthocyanins in the respective T-DNA-inserted mutants. The functional genomics approach through the integration of metabolomics and transcriptomics presented here provides an innovative means of identifying novel gene functions involved in plant metabolism.

Arabidopsis↗

Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1;1, a cytosolic glutamine synthetase1;1.

Rice (Oryza sativa L.) plants possess three homologous but distinct genes for cytosolic glutamine synthetase (GS1): these are OsGS1;1, OsGS1;2, and OsGS1;3. OsGS1;1 was expressed in all organs tested with higher expression in leaf blades, while OsGS1;2, and OsGS1;3 were expressed mainly in roots and spikelets, respectively. We characterized knockout mutants caused by insertion of endogenous retrotransposon Tos17 into the exon-8 (lines ND8037 and ND9801) or the exon-10 (line NC2327) of OsGS1;1. Mendelian segregation occurred in each progeny. Homozygously inserted mutants showed severe retardation in growth rate and grain filling when grown at normal nitrogen concentrations. Abnormal mRNA for GS1;1 was transcribed, and the GS1 protein and its activity in the leaf blades were barely detectable in these mutants. The glutamine pool in the roots and leaf blades of the mutants was lower than that of the wild type. Re-introduction of OsGS1;1 cDNA under the control of its own promoter into the mutants successfully complemented these phenotypes. Progeny where Tos17 was heterozygously inserted or deleted during segregation showed normal phenotypes. The results indicate that GS1;1 is important for normal growth and grain filling in rice; GS1;2 and GS1;3 were not able to compensate for GS1;1 function.

Gene Expression Profiling↗

Passive immunization of the Abeta42(43) C-terminal-specific antibody BC05 in a mouse model of Alzheimer's disease.

BACKGROUND: Over the past few years, amyloid beta protein (Abeta) vaccination has become one of the most effective treatments for Alzheimer's disease. However, the appearance of severe side effects during clinical trials has highlighted the need for improved safety and efficacy. Although antibodies directed against the amino (N)-termini of Abeta are highly effective for passive immunization, a substantial risk of inducing cerebral hemorrhage has been documented. OBJECTIVE: We investigated the effect of the administration of BC05, which was the first antibody developed against the carboxyl (C)-termini of Abeta42(43), on the clearance of brain Abeta42(43). METHOD: The BC05 antibody was injected into the peritoneal cavity of Tg2576 transgenic mice expressing betaAPP(KM670/671NL) once a week from 3 to 12 months of age. RESULTS: BC05 caused a selective 44-fold increase in plasma Abeta42(43) and a significant increase in brain soluble Abeta42(43), showing a 156% difference. Brain insoluble Abeta40 and Abeta42(43) levels were decreased by 27.3 and 31.5%, respectively. A reduction in the number of BAN50-labeled plaques was observed. CONCLUSIONS: BC05 might render Abeta42(43) soluble within the brain and inhibit the insoluble deposition of Abeta40 and Abeta42(43). By analyzing the mechanism of the elevation of soluble Abeta42(43) after passive immunization of BC05, safer and more effective methods of immunotherapy for Alzheimer's disease might be developed.

Alzheimer Disease↗