Junko Shibato

MALAT‐1 enhances cell motility of lung adenocarcinoma cells by influencing the expression of motility‐related genes

MALAT‐1, a long non‐coding RNA, is associated with metastasis, but its role in the metastatic process remains unknown. Here, we show that short‐interfering RNA‐mediated MALAT‐1 silencing impaired in vitro cell motility of lung cancer cells and influenced the expression of numerous genes. In these genes, knockdown of any one of CTHRC1, CCT4, HMMR, or ROD1 clearly inhibited cell migration. In MALAT‐1 knockdown cells, pre‐mRNA levels were decreased in some but not all genes. Thus, our findings suggest that MALAT‐1 is a novel class of non‐coding RNA that promotes cell motility through transcriptional and post‐transcriptional regulation of motility related gene expression.

Physiology and proteomics of the water-deficit stress response in three contrasting peanut genotypes.

Peanut genotypes from the US mini-core collection were analysed for changes in leaf proteins during reproductive stage growth under water-deficit stress. One- and two-dimensional gel electrophoresis (1- and 2-DGE) was performed on soluble protein extracts of selected tolerant and susceptible genotypes. A total of 102 protein bands/spots were analysed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis. Forty-nine non-redundant proteins were identified, implicating a variety of stress response mechanisms in peanut. Lipoxygenase and 1l-myo-inositol-1-phosphate synthase, which aid in inter- and intracellular stress signalling, were more abundant in tolerant genotypes under water-deficit stress. Acetyl-CoA carboxylase, a key enzyme of lipid biosynthesis, increased in relative abundance along with a corresponding increase in epicuticular wax content in the tolerant genotype, suggesting an additional mechanism for water conservation and stress tolerance. Additionally, there was a marked decrease in the abundance of several photosynthetic proteins in the tolerant genotype, along with a concomitant decrease in net photosynthesis in response to water-deficit stress. Differential regulation of leaf proteins involved in a variety of cellular functions (e.g. cell wall strengthening, signal transduction, energy metabolism, cellular detoxification and gene regulation) indicates that these molecules could affect the molecular mechanism of water-deficit stress tolerance in peanut.

Novel rice OsSIPK is a multiple stress responsive MAPK family member showing rhythmic expression at mRNA level

We report isolation and transcriptional profiling of rice (Oryza sativa L.) mitogen-activated protein kinase (MAPK), OsSIPK (salicylic acid-induced protein kinase). OsSIPK gene is located on chromosome 6 most probably existing as a single copy in the rice genome, and encodes 398 amino acid polypeptide having the MAPK family signature and phosphorylation activation motif TEY. Steady state mRNA analyses of OsSIPK showed weak constitutive expression in leaves of 2-week-old rice seedlings. A time course (30–120xa0min) experiment using a variety of elicitors and stresses revealed that the OsSIPK mRNA is strongly induced by jasmonic acid (JA), salicylic acid (SA), ethephon, abscisic acid, cycloheximide (CHX), JA/SAxa0+xa0CHX, cantharidin, okadaic acid, hydrogen peroxide, chitosan, sodium chloride, and cold stress (12°C), but not with wounding by cut, gaseous pollutants ozone, and sulfur dioxide, high temperature, ultraviolet C irradiation, sucrose, and drought. Its transcription was also found to be tissue-specifically regulated, and followed a rhythmic dark induction in leaves. Finally, we showed that the OsSIPK protein is localized to the nucleus. From these results, OsSIPK can be implicated in diverse stimuli-responsive signaling cascades and transcription of certain genes.

Using metabolic profiling to assess plant-pathogen interactions: an example using rice (Oryza sativa) and the blast pathogen Magnaporthe grisea

A metabolomics based approach has been used to study the infection of the Hwacheong rice cultivar (Oryza sativa L. cv. Hwacheong) with compatible (KJ201) and incompatible (KJ401) strains of the rice blast fungal pathogen Magnaporthe grisea. The metabolic response of the rice plants to each strain was assessed 0, 6, 12, 24, 36, and 48xa0h post inoculation. Nuclear Magnetic Resonance (NMR) spectroscopy and Gas and Liquid Chromatography Tandem Mass spectrometry (GC/LC-MS/MS) were used to study both aqueous and organic phase metabolites, collectively resulting in the identification of 93 compounds. Clear metabolic profiles were observed at each time point but there were no significant differences in the metabolic response elicited by each pathogen strain until 24xa0h post inoculation. The largest change was found to be in alanine, which was ~30% (±9%) higher in the leaves from the compatible, compared to the resistant, plants. Together with several other metabolites (malate, glutamine, proline, cinnamate and an unknown sugar) alanine exhibited a good correlation between time of fungal penetration into the leaf and the divergence of metabolite profiles in each interaction. The results indicate both that a wide range of metabolites can be identified in rice leaves and that metabolomics has potential for the study of biochemical changes in plant-pathogen interactions.

Omic analyses unravels global molecular changes in the brain and liver of a rat model for chronic sake (Japanese alcoholic beverage) intake

The effects of chronic administration of Sake (Japanese alcoholic beverage, Nihonshu) on brain and liver of female F334 (Fisher) rats were surveyed via global omic analyses using DNA microarray, 2‐DE, and proton nuclear magnetic resonance. Rats weaned at 4u2009wk of age were given free access to Sake (15% alcohol), instead of water. At 13 months of age, and 24u2009h after withdrawal of Sake supply, rats were sacrificed, and the whole brain and liver tissues dissected for analyses. In general, molecular changes in brain were found to be less than those in liver. Transcriptomics data revealed 36 and 9, and 80 and 62 up‐ and down‐regulated genes, in the brain and liver, respectively, with binding and catalytic activity gene categories the most prominently changed. Results suggested Sake‐induced fragility of brain and liver toxicity/damage, though no significant abnormalities in growth were seen. At protein level, a striking decrease was found in the expression of NADH dehydrogenase (ubiquinone) Fe‐S protein 1 in brain, suggesting attenuation of mitochondrial metabolism. In liver, results again suggested an attenuation of mitochondrial function and, in addition, glycoproteins with unknown function were induced at protein and gene levels, suggesting possible changes in glycoprotein binding in that organ. Metabolomic analysis of brain revealed significant increases in valine, arginine/ornithine, alanine, glutamine, and choline with decreases in isoleucine, N‐acetyl aspartate, taurine, glutamate, and gamma aminobutyric acid. Our results provide a detailed inventory of molecular components of both brain and liver after Sake intake, and may help to better understand effects of chronic Sake drinking.

Gel-based proteomics approach for detecting low nitrogen-responsive proteins in cultivated rice species

Nitrogen fertilization is essential for increasing rice production to meet the food demands of increasing world’s population. We established an in vivo hydroponic rice seedling culture system to investigate physio-biochemical/molecular responses of various rice japonica and indica cultivars to low nitrogen (N). Three-week-old seedlings grown in Yoshida’s nutrient solution manifested stable and reproducible symptoms, such as reduced shoot growth and length under low N. Out of 12 genetically selected cultivars, 11 cultivars showed varied degrees of growth reduction response to applied N (4 and 40 ppm N for treatment and control, respectively), whereas one cultivar (no. 12) showed similar growth as the control though its leaf width was smaller than control. Leaves of a representative low N-responsive cultivar (BG90-2) were sampled for revealing protein profiles between low and normal (control) N application by two-dimensional gel electrophoresis (2-DGE). Forty-one proteins were identified with MALDI-TOF-MS and nESI-LC-MS/MS. Assignment of proteins into major (energy metabolism, photosynthesis and oxidative stress) and minor functional categories, revealed many novel low N-responsive proteins, including those having energy/photosynthesis- and defense/stress- and iron homeostasis-related functions. Results suggest the usefulness of proteomics in identifying novel N-responsive proteins and may provide potential markers for rice response to low N.

The Arabidopsis aminopeptidase LAP2 regulates plant growth, leaf longevity and stress response

Peptidases are known to play key roles in multiple biological processes in all living organisms. In higher plants, the vast majority of putative aminopeptidases remain uncharacterized. In this study, we performed functional and expression analyses of the Arabidopsis LAP2 through cDNA cloning, isolation of T-DNA insertional mutants, characterization of the enzymatic activity, characterization of gene expression and transcriptomics and metabolomics analyses of the mutants. Loss of function of LAP2, one of the 28 aminopeptidases in Arabidopsis, reduced vegetative growth, accelerated leaf senescence and rendered plants more sensitive to various stresses. LAP2 is highly expressed in the leaf vascular tissue and the quiescent center region. Integration of global gene expression and metabolite analyses suggest that LAP2 controlled intracellular amino acid turnover. The mutant maintained free leucine by up-regulating key genes for leucine biosynthesis. However, this influenced the flux of glutamate strikingly. As a result, γ-aminobutyric acid, a metabolite that is derived from glutamate, was diminished in the mutant. Decrements in these nitrogen-rich compounds are associated with morphological alterations and stress sensitivity of the mutant. The results indicate that LAP2 is indeed an enzymatically active aminopeptidase and plays key roles in senescence, stress response and amino acid turnover.

Transcriptomic analysis of rat brain tissue following gamma knife surgery: Early and distinct bilateral effects in the un-irradiated striatum

Gamma knife surgery (GKS) is used for the treatment of various human brain disorders. However, the biological effects of gamma ray irradiation on both the target area, and the surrounding tissues are not well studied. The effects of gamma ray exposure to both targeted and untargeted regions were therefore evaluated by monitoring gene expression changes in the unilateral irradiated (60 Gy) and contralateral un-irradiated striata in the rat. Striata of irradiated and control brains were dissected 16 hours post-irradiation for analysis using a whole genome 44K DNA oligo microarray approach. The results revealed 230 induced and 144 repressed genes in the irradiated striatum and 432 induced and 239 repressed genes in the un-irradiated striatum. Out of these altered genes 39 of the induced and 16 of the reduced genes were common to both irradiated and un-irradiated tissue. Results of semiquantitative, confirmatory RT-PCR and western blot analyses suggested that γ-irradiation caused cellular damage, including oxidative stress, in the striata of both hemispheres of the brains of treated animals.

2D-DIGE-based proteome expression changes in leaves of rice seedlings exposed to low-level gamma radiation at Iitate village, Fukushima

The present study continues our previous research on investigating the biological effects of low-level gamma radiation in rice at the heavily contaminated Iitate village in Fukushima, by extending the experiments to unraveling the leaf proteome. 14-days-old plants of Japonica rice (Oryza sativa L. cv. Nipponbare) were subjected to gamma radiation level of upto 4 µSv/h, for 72 h. Following exposure, leaf samples were taken from the around 190 µSv/3 d exposed seedling and total proteins were extracted. The gamma irradiated leaf and control leaf (harvested at the start of the experiment) protein lysates were used in a 2-D differential gel electrophoresis (2D-DIGE) experiment using CyDye labeling in order to asses which spots were differentially represented, a novelty of the study. 2D-DIGE analysis revealed 91 spots with significantly different expression between samples (60 positive, 31 negative). MALDI-TOF and TOF/TOF mass spectrometry analyses revealed those as comprising of 59 different proteins (50 up-accumulated, 9 down-accumulated). The identified proteins were subdivided into 10 categories, according to their biological function, which indicated that the majority of the differentially expressed proteins consisted of the general (non-energy) metabolism and stress response categories. Proteome-wide data point to some effects of low-level gamma radiation exposure on the metabolism of rice leaves.

ADHD animal model characterization: transcriptomics and proteomics analyses.

Mechanisms underlying behavioral abnormalities of patients with attention-deficit hyperactivity disorder (ADHD) disorder are still unknown. It is worth clarifying alterations in the brain of animal models for ADHD. The animals with neonatal treatment with 6-hydroxydopamine (6-OHDA) and congenic wiggling (Wig) rats show motor hyperactivities during a period of darkness at 4 weeks of age. In rats with 6-OHDA lesions, subcutaneous injection of methamphetamine attenuates hyperactivity, the reverse of its effect in Wig rats. To understand mechanisms underlying such behavioral abnormalities, transcriptomics and proteomics analyses may provide novel information in brain research. The expression of genes and proteins in brain regions can be measured by DNA microarray and two-dimensional gel electrophoresis, respectively. We have shown different expressions of genes and proteins in brains of rats with neonatal 6-OHDA lesions and Wig rats.