Biomedical subjects

Junko Shibato

Publications and source records attributed to Junko Shibato.

11 recordsLinked to original sources

PubMed2006

New protein extraction/solubilization protocol for gel-based proteomics of rat (female) whole brain and brain regions.

The rat is an accepted model for studying human psychiatric/neurological disorders. We provide a protocol for total soluble protein extraction using trichloroacetic acid/acetone (TCA/A) from rat (female) whole brain, 10 brain regions and the pituitary gland, and show that two-dimensional gel electrophoresis (2-DGE) using pre-cast immobilized pH (4-7) gradient (IPG) strip gels (13 cm) in the first dimension yields clean silver nitrate stained protein profiles. Though TCA/A precipitation may not be "ideal", the important choice here is the selection of an appropriate lysis buffer (LB) for solubilizing precipitated proteins. Our results reveal enrichment of protein spots by use of individual brain regions rather than whole brain, as well as the presence of differentially expressed spots in their proteomes. Thus individual brain regions provide improved protein coverage and are better suited for differential protein detection. Moreover, using a phosphoprotein-specific dye, in-gel detection of phosphoproteins was demonstrated. Representative high-resolution silver nitrate stained proteome profiles of rat whole brain total soluble protein are presented. Shortcomings apart (failure to separate membrane proteins), gel-based proteomics remains a viable option, and 2-DGE is the method of choice for generating high-resolution proteome maps of rat brain and brain regions.

Animals↗

PubMed2006

DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and gamma-rays.

Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma (gamma)-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and gamma-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and gamma-rays). Similarly, for X- and gamma-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and gamma-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-à-vis their energy levels.

Down-Regulation↗

PubMed2006

Effect of textile wastewaters on Saccharomyces cerevisiae using DNA microarray as a tool for genome-wide transcriptomics analysis.

Textile mill effluents (TMEs) discharged from the textile industry can be considered as one class of hypothetical toxicants in the environment. To investigate the potential toxicity of TMEs, we applied cDNA microarray technology to examine the genome-wide expression profiles in model eukaryote, Saccharomyces cerevisiae. The results revealed a rich source of genetic information for the yeast cells that were exposed to the untreated and treated TMEs. Among the 5956 valid genes, 275 genes were up-regulated and 40 genes were down-regulated for the untreated TMEs. On the other hand, only 90 genes were up-regulated, and 29 genes were down-regulated upon exposure to the treated TMEs. The changes in gene expression were also confirmed by RT-PCR. The potent up- and down-regulation of genes suggest that yeast cells undergo genome-wide changes in mRNA expression, indicative of a stress response. Additionally, a classification into specific functional gene categories indicated that untreated and even treated TMEs still had toxicity. Especially, the genes related to oxidative stress, such as AHP1, ATX1, GRX1, TRX1 and TRX2, were up-regulated in treated TMEs that can directly reach to surface and ground waters, and sediments.

Down-Regulation↗

PubMed2006

Functional characterization of OsRacB GTPase--a potentially negative regulator of basal disease resistance in rice.

The rice genome contains at least seven expressed Rop small GTPase genes. Of these Rops, OsRac1 is the only characterized gene that has been implicated in disease resistance as a positive regulator. To our interest in finding a negative ROP regulator of disease resistance in rice, we applied a "phylogeny of function" approach to rice Rops, and identified OsRacB based on its close genetic orthologous relationship with the barley HvRacB gene, a known negative regulator of disease resistance. To determine the function of OsRacB, we isolated the OsRacB cDNA and conducted gene expression and transgenic studies. OsRacB, a single copy gene in the genome of rice, shared 98% identity with HvRacB at the amino acid level. Its mRNA was strongly expressed in leaf sheath (LS) and in panicles, but was very weakly expressed in young and mature leaves. The basal mRNA level of OsRacB in LS of two-week-old seedlings was strongly down-regulated upon wounding by cut and treatment with jasmonic acid. A dramatic down-regulation in the OsRacB transcripts was also found in plants inoculated with the blast pathogen, Magnaporthe grisea. Interestingly, transgenic rice plants over-expressing OsRacB showed increased symptom development in response to rice blast pathogens. Additionally, fluorescence microscopy of green fluorescent protein (GFP):OsRacB-transformed onion cells and Arabidopsis protoplasts revealed OsRacB association with plasma membrane (PM), suggesting that PM localization is required for proper function of OsRacB. Based on these results, we suggest that OsRacB functions as a potential regulator for a basal disease resistance pathway in rice.

Amino Acid Sequence↗

PubMed2006

Differential expression of defense/stress-related marker proteins in leaves of a unique rice blast lesion mimic mutant (blm).

We analyzed a unique rice (Oryza sativa L.) blast lesion mimic (blm) mutant for differentially expressed proteins in leaves of one- and two-week-old seedlings manifesting the lesion mimic phenotype. Gel-based one- and two-dimensional electrophoresis (1- and 2-DGE) was performed using leaves (blm and wild-type, WT) before (stage 1, S1) and after (stage 2, S2) lesion formation. 1-DGE immunoblotting revealed potent increase in the expression of a key pathogenesis-related (PR) marker biosynthetic enzyme, naringenin 7-O-methyltransferase, involved in rice phytoalexin sakuranetin biosynthesis, and three oxidative-stress-related marker proteins, catalase, ascorbate peroxidase (APX), and superoxide dismutase (SOD) in leaves of the blm mutant. 2-D gel immunoblotting analysis with anti-APX and anti-SOD antibodies revealed newly appearing cross-reacting protein spots in blm. 2-DGE analysis detected 50 Coomassie brilliant blue-stained protein spots differentially expressed in blm. A total of 23 and 44 protein spots was excised for analysis by N-terminal amino acid sequencing and nano-electrospray ionization liquid chromatography mass spectrometry, respectively; 26 nonredundant proteins were identified. The pathogenesis-related class 5 and 10 proteins, including a new OsPR10d protein, were significantly induced in blm. The OsPR5 protein spot was stained with Pro-Q Diamond phosphoprotein gel stain suggesting OsPR5 to be a putative phosphoprotein. Surprisingly, protein spot 20, a leaf OsPR10b, showed identity to a rice root-specific PR-10 (RSOsPR10). To resolve this discrepancy, we checked its expression in leaves of blm and WT (S1 and S2), respectively, using gene-specific primers and reverse transcriptase-polymerase chain reaction; RSOsPR10 mRNA was found to express in the leaves.

Amino Acid Sequence↗

PubMed2005

A hydroponic rice seedling culture model system for investigating proteome of salt stress in rice leaf.

By using an in vivo hydroponic rice seedling culture system, we investigated the physiological and biochemical responses of a model rice japonica cultivar Nipponbare to salt stress using proteomics and classical biochemical methods. Yoshida's nutrient solution (YS) was used to grow rice seedlings. YS-grown 18-day-old seedlings manifested highly stable and reproducible symptoms, prominently the wilting and browning of the 3rd leaf, reduced photosynthetic activity, inhibition in overall seedling growth, and failure to develop new (5th) leaf, when subjected to salt stress by transferring them to YS containing 130 mM NaCl for 4 days. As leaf response to salt stress is least investigated in rice by proteomics, we used the 3rd leaf as source material. A comparison of 2-DE protein profiles between the untreated control and salt-stressed 3rd leaves revealed 55 differentially expressed CBB-stained spots, where 47 spots were increased over the control. Of these changed spots, the identity of 33 protein spots (27 increased and 5 decreased) was determined by nESI-LC-MS/MS. Most of these identified proteins belonged to major metabolic processes like photosynthetic carbon dioxide assimilation and photorespiration, suggesting a good correlation between salt stress-responsive proteins and leaf morphology. Moreover, 2-DE immunoblot and enzymatic activity analyses of 3rd leaves revealed remarkable changes in the key marker enzymes associated with oxidative damage to salt stress: ascorbate peroxidase and lipid peroxidation were induced, and catalase was suppressed. These results demonstrate that hydroponic culture system is best suited for proteomics of salt stress in rice seedling.

Amino Acid Sequence↗

PubMed2003

Diverse environmental cues transiently regulate OsOPR1 of the "octadecanoid pathway" revealing its importance in rice defense/stress and development.

Previously, we reported rice (Oryza sativa L. japonica type cv. Nipponbare) allene oxide synthase (OsAOS) and cyclase (OsAOC) genes of the octadecanoid pathway. Here we have isolated a 12-oxo-phytodienoic acid reductase gene, called OsOPR1, encoding the last committed enzymatic step on the octadecanoid pathway leading to jasmonic acid (JA) biosynthesis. OsOPR1 encodes a 380 amino acid long polypeptide with a predicted molecular mass of 42465.02 and pI of 5.79, and belongs to a gene family in the rice genome. Transcriptional profiling using our established two-week-old rice seedling model system against a variety of environmental factors such as wounding, global signals (including JA), osmotic stresses, heavy metals, UV-C irradiation, fungal elicitor, protein phosphatase inhibitors, and gaseous pollutants indicated that OsOPR1 transcript was rapidly, transiently and differentially up-regulated within 30 min in leaves. Surprisingly, co-application of signaling molecules JA, salicylate and ethylene, resulted in a massive accumulation of the OsOPR1 transcript at 30 min and remained elevated with time, a new observation. Furthermore, transient expression of OsOPR1, most likely regulated by a de novo synthesized negative trans-acting factor(s), was evidenced by the use of cycloheximide. Finally, the endogenous OsOPR1 expression varied with the stage of plant development. These results strongly suggest a regulatory role for OsOPR1 in rice plant defense/stress response pathway(s) and reproduction.

Amino Acid Sequence↗

PubMed2003

Damage of yeast cells induced by pulsed light irradiation.

DNA damage, such as formation of single strand breaks and pyrimidine dimers was induced in yeast cells after irradiation by pulsed light, which were essentially the same as observed with continuous ultraviolet (UV) light. The UV-induced DNA damage is slightly higher than seen with pulsed light. However, increased concentration of eluted protein and structural change in the irradiated yeast cells were observed only in the case of pulsed light. A difference in the inactivation effect between pulsed light and UV light was found and this suggested cell membrane damage induced by pulsed light irradiation. It is proposed that pulsed light can be used as an effective sterilizing method for the yeast Saccharomyces cerevisiae.

Consumer Product Safety↗

PubMed2003

Novel rice MAP kinases OsMSRMK3 and OsWJUMK1 involved in encountering diverse environmental stresses and developmental regulation.

We report isolation of two novel rice (Oryza sativa L.) mitogen-activated protein kinases (MAPKs), OsMSRMK3 (multiple stress responsive) and OsWJUMK1 (wound- and JA-uninducible) that most likely exist as single copy genes in its genome. OsMSRMK3 and OsWJUMK1 encode 369 and 569 amino acid polypeptides having the MAPK family signature and phosphorylation activation motifs TEY and TDY, respectively. Steady state mRNA analyses of these MAPKs with constitutive expression in leaves of two-week-old seedlings revealed that OsMSRMK3 was up-regulated upon wounding (by cut), jasmonic acid (JA), salicylic acid (SA), ethylene, abscisic acid, hydrogen peroxide (H(2)O(2)), protein phosphatase inhibitors, chitosan, high salt/sugar, and heavy metals, whereas OsWJUMK1 not induced by either wounding, JA or SA, showed up-regulation only by H(2)O(2), heavy metals, and cold stress (12 degrees C). Moreover, these MAPKs were developmentally regulated. These results strongly suggest a role for OsMSRMK3 and OsWJUMK1 in both stress-signalling pathways and development in rice.

Amino Acid Sequence↗

PubMed2002

The curved DNA structure in the 5'-upstream region of the light-responsive genes: its universality, binding factor and function for cyanobacterial psbA transcription.

A unique DNA curvature, the CIT, has been found in the 5'-upstream region of the psbA2 gene, which exhibits basal, light-responsive and circadian rhythmic transcription, in a unicellular photosynthetic cyanobacterium, Microcystis aeruginosa K-81. In this study, we report the universality of curvatures found in 5'-upstream regions in the psbA family and the function of the curvature in gene expression. Intrinsic curvatures were identified within 1000 bp upstream from the psbA genes in another cyanobacterium, a red alga and in plants (monocot and dicot). Mutagenized curvatures were constructed and confirmed to have disrupted architecture by gel electrophoresis and atomic force microscopy. Relatively small amounts but light-responsive transcripts of psbA2 were observed in cyanobacterial transformants harboring the mutagenized curvature under light/dark and light/high-light conditions. This shows that the curvature is important for basal transcription. In vitro primer extension and DNA mobility shift assay revealed that factors which might bind to the region upstream from the bending center contribute to the effective basal transcription of psbA2.

5' Flanking Region↗

PubMed2002

Biochemical and genetic characterization of mundticin KS, an antilisterial peptide produced by Enterococcus mundtii NFRI 7393.

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.

Amino Acid Sequence↗