Showing papers by "University of Tsukuba published in 2002"

Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray.

Abstract: Full-length cDNAs are essential for functional analysis of plant genes in the post-sequencing era of the Arabidopsis genome. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. We have prepared a full-length cDNA microarray containing approximately 7000 independent, full-length cDNA groups to analyse the expression profiles of genes under drought, cold (low temperature) and high-salinity stress conditions over time. The transcripts of 53, 277 and 194 genes increased after cold, drought and high-salinity treatments, respectively, more than fivefold compared with the control genes. We also identified many highly drought-, cold- or high-salinity- stress-inducible genes. However, we observed strong relationships in the expression of these stress-responsive genes based on Venn diagram analysis, and found 22 stress-inducible genes that responded to all three stresses. Several gene groups showing different expression profiles were identified by analysis of their expression patterns during stress-responsive gene induction. The cold-inducible genes were classified into at least two gene groups from their expression profiles. DREB1A was included in a group whose expression peaked at 2 h after cold treatment. Among the drought, cold or high-salinity stress-inducible genes identified, we found 40 transcription factor genes (corresponding to approximately 11% of all stress-inducible genes identified), suggesting that various transcriptional regulatory mechanisms function in the drought, cold or high-salinity stress signal transduction pathways.

Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants

Abstract: Coordinate induction of phase 2 proteins and elevation of glutathione protect cells against the toxic and carcinogenic effects of electrophiles and oxidants. All inducers react covalently with thiols at rates that are closely related to their potencies. Inducers disrupt the cytoplasmic complex between the actin-bound protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2 to migrate to the nucleus where it activates the antioxidant response element (ARE) of phase 2 genes and accelerates their transcription. We cloned, overexpressed, and purified murine Keap1 and demonstrated on native gels the formation of complexes of Keap1 with the Neh2 domain of Nrf2 and their concentration-dependent disruption by inducers such as sulforaphane and bis(2-hydroxybenzylidene)acetone. The kinetics, stoichiometry, and order of reactivities of the most reactive of the 25 cysteine thiol groups of Keap1 have been determined by tritium incorporation from [3H]dexamethasone mesylate (an inducer and irreversible modifier of thiols) and by UV spectroscopy with sulforaphane, 2,2′-dipyridyl disulfide and 4,4′-dipyridyl disulfide (titrants of thiol groups), and two closely related Michael reaction acceptors [bis(2- and 4-hydroxybenzylidene)acetones] that differ 100-fold in inducer potency and the UV spectra of which are bleached by thiol addition. With large excesses of these reagents nearly all thiols of Keap1 react, but sequential reaction with three successive single equivalents (per cysteine residue) of dipyridyl disulfides revealed excellent agreement with pseudo-first order kinetics, rapid successive declines in reaction velocity, and the stoichiometric formation of two equivalents of thiopyridone per reacted cysteine. This finding suggests that reaction of cysteine thiols is followed by rapid formation of protein disulfide linkages. The most reactive residues of Keap1 (C257, C273, C288, and C297) were identified by mapping the dexamethasone-modified cysteines by mass spectrometry of tryptic peptides. These residues are located in the intervening region between BTB and Kelch repeat domains of Keap1 and probably are the direct sensors of inducers of the phase 2 system.

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs

Abstract: Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences These are clustered into 33,409 'transcriptional units', contributing 901% of a newly established mouse transcriptome database Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome 41% of all transcriptional units showed evidence of alternative splicing In protein-coding transcripts, 79% of splice variations altered the protein product Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics

Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana.

Abstract: Raffinose family oligosaccharides (RFO) accumulating during seed development are thought to play a role in the desiccation tolerance of seeds. However, the functions of RFO in desiccation tolerance have not been elucidated. Here we examine the functions of RFO in Arabidopsis thaliana plants under drought- and cold-stress conditions, based on the analyses of function and expression of genes involved in RFO biosynthesis. Sugar analysis showed that drought-, high salinity- and cold-treated Arabidopsis plants accumulate a large amount of raffinose and galactinol, but not stachyose. Raffinose and galactinol were not detected in unstressed plants. This suggests that raffinose and galactinol are involved in tolerance to drought, high salinity and cold stresses. Galactinol synthase (GolS) catalyses the first step in the biosynthesis of RFO from UDP-galactose. We identified three stress-responsive GolS genes (AtGolS1, 2 and 3) among seven Arabidopsis GolS genes. AtGolS1 and 2 were induced by drought and high-salinity stresses, but not by cold stress. By contrast, AtGolS3 was induced by cold stress but not by drought or salt stress. All the GST fusion proteins of GST-AtGolS1, 2 and 3 expressed in Escherichia coli had galactinol synthase activities. Overexpression of AtGolS2 in transgenic Arabidopsis caused an increase in endogenous galactinol and raffinose, and showed reduced transpiration from leaves to improve drought tolerance. These results show that stress-inducible galactinol synthase plays a key role in the accumulation of galactinol and raffinose under abiotic stress conditions, and that galactinol and raffinose may function as osmoprotectants in drought-stress tolerance of plants.

Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater

Abstract: Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium that causes life-threatening gas gangrene and mild enterotoxaemia in humans, although it colonizes as normal intestinal flora of humans and animals. The organism is known to produce a variety of toxins and enzymes that are responsible for the severe myonecrotic lesions. Here we report the complete 3,031,430-bp sequence of C. perfringens strain 13 that comprises 2,660 protein coding regions and 10 rRNA genes, showing pronounced low overall G + C content (28.6%). The genome contains typical anaerobic fermentation enzymes leading to gas production but no enzymes for the tricarboxylic acid cycle or respiratory chain. Various saccharolytic enzymes were found, but many enzymes for amino acid biosynthesis were lacking in the genome. Twenty genes were newly identified as putative virulence factors of C. perfringens, and we found a total of five hyaluronidase genes that will also contribute to virulence. The genome analysis also proved an efficient method for finding four members of the two-component VirR/VirS regulon that coordinately regulates the pathogenicity of C. perfringens. Clearly, C. perfringens obtains various essential materials from the host by producing several degradative enzymes and toxins, resulting in massive destruction of the host tissues.

Functional annotation of a full-length Arabidopsis cDNA collection.

Abstract: Full-length complementary DNAs (cDNAs) are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We isolated 155,144 RIKEN Arabidopsis full-length (RAFL) cDNA clones. The 3'-end expressed sequence tags (ESTs) of 155,144 RAFL cDNAs were clustered into 14,668 nonredundant cDNA groups, about 60% of predicted genes. We also obtained 5' ESTs from 14,034 nonredundant cDNA groups and constructed a promoter database. The sequence database of the RAFL cDNAs is useful for promoter analysis and correct annotation of predicted transcription units and gene products. Furthermore, the full-length cDNAs are useful resources for analyses of the expression profiles, functions, and structures of plant proteins.

Hemoprotein Bach1 regulates enhancer availability of heme oxygenase‐1 gene

Abstract: Heme oxygenase-1 (HO-1) protects cells from various insults including oxidative stress. Transcriptional activators, including the Nrf2/Maf heterodimer, have been the focus of studies on the inducible expression of ho-1. Here we show that a heme-binding factor, Bach1, is a critical physiological repressor of ho-1. Bach1 bound to the multiple Maf recognition elements (MAREs) of ho-1 enhancers with MafK in vitro and repressed their activity in vivo, while heme abrogated this repressor function of Bach1 by inhibiting its binding to the ho-1 enhancers. Gene targeting experiments in mice revealed that, in the absence of Bach1, ho-1 became expressed constitutively at high levels in various tissues under normal physiological conditions. By analyzing bach1/nrf2 compound-deficient mice, we documented antagonistic activities of Bach1 and Nrf2 in several tissues. Chromatin immunoprecipitation revealed that small Maf proteins participate in both repression and activation of ho-1. Thus, regulation of ho-1 involves a direct sensing of heme levels by Bach1 (by analogy to lac repressor sensitivity to lactose), generating a simple feedback loop whereby the substrate effects repressor–activator antagonism.

HAR1 mediates systemic regulation of symbiotic organ development

Abstract: Symbiotic root nodules are beneficial to leguminous host plants; however, excessive nodulation damages the host because it interferes with the distribution of nutrients in the plant. To keep a steady balance, the nodulation programme is regulated systemically in leguminous hosts. Leguminous mutants that have lost this ability display a hypernodulating phenotype. Through the use of reciprocal and self-grafting studies using Lotus japonicus hypernodulating mutants, har1 (also known as sym78), we show that the shoot genotype is responsible for the negative regulation of nodule development. A map-based cloning strategy revealed that HAR1 encodes a protein with a relative molecular mass of 108,000, which contains 21 leucine-rich repeats, a single transmembrane domain and serine/threonine kinase domains. The har1 mutant phenotype was rescued by transfection of the HAR1 gene. In a comparison of Arabidopsis receptor-like kinases, HAR1 showed the highest level of similarity with CLAVATA1 (CLV1). CLV1 negatively regulates formation of the shoot and floral meristems through cell-cell communication involving the CLV3 peptide. Identification of hypernodulation genes thus indicates that genes in leguminous plants bearing a close resemblance to CLV1 regulate nodule development systemically, by means of organ-organ communication.

Chloroplast avoidance movement reduces photodamage in plants

Abstract: When plants are exposed to light levels higher than those required for photosynthesis, reactive oxygen species are generated in the chloroplasts and cause photodamage. This can occur even under natural growth conditions. To mitigate photodamage, plants have developed several protective mechanisms. One is chloroplast avoidance movement, in which chloroplasts move from the cell surface to the side walls of cells under high light conditions, although experimental support is still awaited. Here, using different classes of mutant defective in chloroplast avoidance movement, we show that these mutants are more susceptible to damage in high light than wild-type plants. Damage of the photosynthetic apparatus and subsequent bleaching of leaf colour and necrosis occur faster under high light conditions in the mutants than in wild-type plants. We conclude that chloroplast avoidance movement actually decreases the amount of light absorption by chloroplasts, and might therefore be important to the survival of plants under natural growth conditions.

Observation of B̄0 → D(*)0 pp̄

Abstract: The B meson decay modes B → Dpp and B → D*pp have been studied using 29.4 fb - 1 of data collected with the Belle detector at KEKB. The B 0 → D 0 pp and B 0 → D* 0 pp decays have been observed for the first time with branching fractions B(B 0 → D 0 pp) = (1.18 ′ 0.15 ′ 0.16) X 10 - 4 and B(B 0 → D* 0 pp) = (1.20 + 0 . 3 3 - 0 . 2 9 ′ 0.21) X 10 - 4 . No signal has been found for the B + → D + pp and B + → D* + pp decay modes, and the corresponding upper limits at 90% C.L. are presented.

A blue-light-activated adenylyl cyclase mediates photoavoidance in Euglena gracilis

Abstract: Blue light regulates processes such as the development of plants and fungi and the behaviour of microbes. Two types of blue-light receptor flavoprotein have been identified: cryptochromes, which have partial similarity to photolyases, and phototropins, which are photoregulated protein kinases. The former have also been found in animals with evidence of essential roles in circadian rhythms. Euglena gracilis, a unicellular flagellate, abruptly changes its swimming direction after a sudden increase or decrease in incident blue light intensity, that is, step-up or step-down photophobic responses, resulting in photoavoidance or photoaccumulation, respectively. Although these photobehaviours of Euglena have been studied for a century, the photoreceptor molecules mediating them have remained unknown. Here we report the discovery and biochemical characterization of a new type of blue-light receptor flavoprotein, photoactivated adenylyl cyclase, in the photoreceptor organelle of Euglena gracilis, with molecular genetic evidence that it mediates the step-up photophobic response.

Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice.

Abstract: Mice that lack the Nrf2 basic-region leucine-zipper transcription factor are more sensitive than wild-type (WT) animals to the cytotoxic and genotoxic effects of foreign chemicals and oxidants. To determine the basis for the decrease in tolerance of the Nrf2 homozygous null mice to xenobiotics, enzyme assay, Western blotting and gene-specific real-time PCR (TaqMan) have been used to examine the extent to which hepatic expression of GSH-dependent enzymes is influenced by the transcription factor. The amounts of protein and mRNA for class Alpha, Mu and Pi glutathione S-transferases were compared between WT and Nrf2 knockout (KO) mice of both sexes under both constitutive and inducible conditions. Among the class Alpha and class Mu transferases, constitutive expression of Gsta1, Gsta2, Gstm1, Gstm2, Gstm3, Gstm4 and Gstm6 subunits was reduced in the livers of Nrf2 mutant mice to between 3% and 60% of that observed in WT mice. Induction of these subunits by butylated hydroxyanisole (BHA) was more marked in WT female mice than in WT male mice. TaqMan analyses showed the increase in transferase mRNA caused by BHA was attenuated in Nrf2(-/-) mice, with the effect being most apparent in the case of Gsta1, Gstm1 and Gstm3. Amongst class Pi transferase subunits, the constitutive hepatic level of mRNA for Gstp1 and Gstp2 was not substantially affected in the KO mice, but their induction by BHA was dependent on Nrf2; this was more obvious in female mutant mice than in male mice. Nrf2 KO mice exhibited reduced constitutive expression of the glutamate cysteine ligase catalytic subunit, and, to a lesser extent, the expression of glutamate cysteine ligase modifier subunit. Little variation was observed in the levels of glutathione synthase in the different mouse lines. Thus the increased sensitivity of Nrf2(-/-) mice to xenobiotics can be partly attributed to a loss in constitutive expression of multiple GSH-dependent enzymes, which causes a reduction in intrinsic detoxification capacity in the KO animal. These data also indicate that attenuated induction of GSH-dependent enzymes in Nrf2(-/-) mice probably accounts for their failure to adapt to chronic exposure to chemical and oxidative stress.

Power Assist System HAL-3 for Gait Disorder Person

Abstract: We have developed the power assistive suit, HAL (Hybrid Assistive Leg) which provide the self-walking aid for gait disorder persons or aged persons In this paper, We introduce HAL-3 system, improving HAL-1,2 systems which had developed previously EMG signal was used as the input information of power assist controller We propose a calibration method to identify parameters which relates the EMG to joint torque by using HAL-3 We could obtain suitable torque estimated by EMG and realize an apparatus that enables power to be used for walking and standing up according to the intention of the operator

Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver

Abstract: Using flow cytometry and single cell-based assays, we prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewing capability. These cells could be clonally propagated in culture where they continuously produced hepatocytes and cholangiocytes as descendants while maintaining primitive stem cells. When cells that expanded in vitro were transplanted into recipient animals, they morphologically and functionally differentiated into hepatocytes and cholangiocytes with reconstitution of hepatocyte and bile duct structures. Furthermore, these cells differentiated into pancreatic ductal and acinar cells or intestinal epithelial cells when transplanted into pancreas or duodenal wall. These data indicate that self-renewing pluripotent stem cells persist in the developing mouse liver and that such cells can be induced to become cells of other organs of endodermal origin under appropriate microenvironment. Manipulation of hepatic stem cells may provide new insight into therapies for diseases of the digestive system.

BCS-BEC crossover in a gas of Fermi atoms with a Feshbach resonance.

Abstract: We discuss the BCS-BEC crossover in a degenerate Fermi gas of two hyperfine states interacting close to a Feshbach resonance. We show that, by including fluctuation contributions to the free energy similar to that considered by Nozieres and Schmitt-Rink, the character of the superfluid phase transition continuously changes from the BCS-type to the BEC-type, as the threshold of the quasimolecular band is lowered. In the BEC regime, the superfluid phase transition is interpreted in terms of molecules associated with both the Feshbach resonance and Cooper pairing.

Identification of the interactive interface and phylogenic conservation of the Nrf2-Keap1 system.

Abstract: Background: The transcription factor Nrf2 and its negative regulator Keap1 play important roles in transcriptional induction of a set of detoxifying and anti-oxidant enzymes. To gain an insight into our present enigma as to how cells receive oxidative and electrophilic signals and transduce them to Nrf2, we have developed a zebrafish model system for molecular toxicological studies. Results: We systematically cloned zebrafish cytoprotective enzyme cDNAs and found their expression to be efficiently induced by electrophilic agents. We consequently identified the presence of Nrf2 and Keap1 in zebrafish. Both loss- and gain-of-function analyses demonstrated that Nrf2 is the primary regulator of a subset of cytoprotective enzyme genes, while Keap1 suppresses Nrf2 activity in zebrafish. An ETGE motif, critical for the Nrf2–Keap1 interaction, was identified in the Neh2 domain of Nrf2 by reverse two-hybrid screening and found to be indispensable for the regulation of Nrf2 activity in zebrafish. Conclusion: Taken together, these results indicate that the Nrf2-Keap1 system is highly conserved among vertebrates and that the interface between Nrf2 and Keap1 forms an important molecular basis of this regulatory system.

Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons

Abstract: Dorsal root ganglion (DRG) neurons specifically project axons to central and peripheral targets according to their sensory modality. The Runt-related genes Runx1 and Runx3 are expressed in DRG neuronal subpopulations, suggesting that they may regulate the trajectories of specific axons. Here we report that Runx3-deficient (Runx3(-/-)) mice displayed severe motor uncoordination and that few DRG neurons synthesized the proprioceptive neuronal marker parvalbumin. Proprioceptive afferent axons failed to project to their targets in the spinal cord as well as those in the muscle. NT-3-responsive Runx3(-/-) DRG neurons showed less neurite outgrowth in vitro. However, we found no changes in the fate specification of Runx3(-/-) DRG neurons or in the number of DRG neurons that expressed trkC. Our data demonstrate that Runx3 is critical in regulating the axonal projections of a specific subpopulation of DRG neurons.

Power assist control for walking aid with HAL-3 based on EMG and impedance adjustment around knee joint

Abstract: This paper describes the power assist control for walking aid based on EMG and impedance adjustment with HAL-3 we have developed. Virtual torque derived from EMG is adopted as a basic control method, and the motion assist control as to operator's intention can be realized by this method. And we suggest the impedance adjustment around knee joint for more effective power assist control. Experiments for simple motion and walking motion were performed to verify the proposed approach, with impedance parameters found by RLS (recursive least square) method. The evaluation of assisted motion was done by a calculation based on EMG in nearly proportion to the operator's muscle force. The results showed the amplitudes of EMG were reduced significantly, the operator was able to swing the leg lighter by reducing the inertia around knee, and the strain of knee in foot-grounding could be alleviated by adding the stiffness to joint.

Birefringence imaging of human skin by polarization-sensitive spectral interferometric optical coherence tomography.

Abstract: We have developed a spectral interferometric optical coherence tomography (OCT) system with polarization sensitivity that is able to measure a two-dimensional tomographic image by means of one-dimensional mechanical scanning. Our system, which has an axial resolution of 32 mum , calculates the distribution of each element of the Muller matrix of a measured object from 16 OCT images. The OCT system successfully reveals the birefringent nature of human skin tissue.