Biomedical subjects

Hitoshi Iwahashi

Publications and source records attributed to Hitoshi Iwahashi.

At least 19 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

Search for novel stress-responsive protein components using a yeast mutant lacking two cytosolic Hsp70 genes, SSA1 and SSA2.

Heat shock proteins (Hsp) 70 are a ubiquitous family of molecular chaperones involved in many cellular processes. A yeast strain, ssa1/2, with two functionally redundant cytosolic Hsp70s (SSA1 and SSA2) deleted shows thermotolerance comparable to mildly heat-shocked wild type yeast, as well as increased protein synthesis and ubiquitin-proteasome protein degradation. Since mRNA abundance does not always correlate well with protein expression levels it is essential to study proteins directly. We used a gel-based approach to identify stress-responsive proteins in the ssa1/2 mutant and identified 43 differentially expressed spots. These were trypsin-digested and analyzed by nano electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). A total of 22 non-redundant proteins were identified, 11 of which were confirmed by N-terminal sequencing. Nine proteins, most of which were up-regulated (2-fold or more) in the ssa1/2 mutant, proved to be stress-inducible proteins such as molecular chaperones and anti-oxidant proteins, or proteins related to carbohydrate metabolism. Interestingly, a translational factor Hyp2p up-regulated in the mutant was also found to be highly phosphorylated. These results indicate that the cytosolic Hsp70s, Ssa1p and Ssa2p, regulate an abundance of proteins mainly involved in stress responses and protein synthesis.

Adenosine Triphosphatases↗

PubMed2006

Role of defense/stress-related marker genes, proteins and secondary metabolites in defining rice self-defense mechanisms.

Rice, a first cereal crop whose draft genome sequence from two subspecies (japonica-type cv. Nipponbare and indica-type 93-11) was available in 2002, along with its almost complete genome sequence in 2005, has drawn the attention of researchers worldwide because of its immense impact on human existence. One of the most critical research areas in rice is to discern the self-defense mechanism(s), an innate property of all living organisms. The last few decades have seen scattered research into rice responses to diverse environmental stimuli and stress factors. Our understanding on rice self-defense mechanism has increased considerably with accelerated research during recent years mainly due to identification and characterization of several defense/stress-related components, genes, proteins and secondary metabolites. As these identified components have been used to study the defense/stress pathways, their compilation in this review will undoubtedly help rice (and others) researchers to effectively use them as a potential marker for better understanding, and ultimately, in defining rice (and plant) self-defense response pathways.

Gene Expression Regulation, Plant↗

PubMed2006

Expression analysis of sex-specific and 17beta-estradiol-responsive genes in the Japanese medaka, Oryzias latipes, using oligonucleotide microarrays.

Gene profiling of Japanese medaka (Oryzias latipes) was performed using an oligonucleotide DNA microarray representing 22,587 TIGR O. latipes gene indices (OLGIs). The average correlation coefficients for gene expression between individual mature fish were high (>0.95) for both female and male, indicating that the physiological status of medaka is highly reproducible under prescribed growth conditions. Of the 22,587 OLGIs, 2575 showed significant differences in expression between female and male. Exposure to 17beta-estradiol (E2) revealed 381 E2-responsive OLGIs in male medaka. Feminization and male-dysfunction factors of the E2-treated males calculated using the combination of Pearson correlation coefficient and Euclidean distances indicate that E2 treatment "weakly feminized" male medaka, while male physiological functions were not significantly disrupted. This study demonstrates the possibility of using medaka microarrays to estimate the overall effects of hormonally active chemicals.

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

Mechanisms of patulin toxicity under conditions that inhibit yeast growth.

Patulin, 4-hydroxy-4H-furo[3,2c]pyran-2(6H)-one, is one of the best characterized and most widely disseminated mycotoxins found in agricultural products. Nonetheless, the mechanisms by which patulin causes toxicity are not well understood. Thus, the cytotoxicity of patulin was characterized by analysis of the yeast transcriptome upon challenge with patulin. Interestingly, patulin-induced yeast gene expression profiles were found to be similar to gene expression patterns obtained after treatment with the antifungal agricultural chemicals thiuram, maneb, and zineb. Moreover, patulin treatment was found to activate protein degradation, especially proteasome activities, sulfur amino acid metabolism, and the defense system for oxidative stress. Damage to DNA by alkylation was also suggested, and this seemed to be repaired by recombinational and excision repair mechanisms. Furthermore, the results provide potential biomarker genes for the detection of patulin in agricultural products. The results suggest the possibility of applying the yeast transcriptome system for the evaluation of chemicals, especially for natural chemicals that are difficult to get by organic synthesis.

Amino Acids, Sulfur↗

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

Plant phosphoproteomics: a long road ahead.

Phosphoproteomics can be defined as the comprehensive study of protein phosphorylation by identification of the phosphoproteins, exact mapping of the phosphorylation sites, quantification of phosphorylation, and eventually, revealing their biological function. Its place in today's research is vitally important to address the most fundamental question - how the phosphorylation events control most, if not all, of the cellular processes in a given organism? Despite the immense importance of phosphorylation, the analysis of phosphoproteins on a proteome-wide scale remains a formidable challenge. Nevertheless, several technologies have been developed, mostly in yeast and mammals, to conduct a large-scale phosphoproteomic study. Some of these technologies have been successfully applied to plants with a few modifications, resulting in documentation of phosphoproteins, phosphorylation site mapping, identification of protein kinase substrates, etc. at the global level. In this review, we summarize in vitro and in vivo approaches for detection and analysis of phosphoproteins including protein kinases and we discuss the importance of phosphoproteomics in understanding plant biology. These approaches along with bioinformatics will help plant researchers to design and apply suitable phosphoproteomic strategies in helping to find answers to their biological questions.

Chromatography↗

PubMed2006

Rejuvenating rice proteomics: facts, challenges, and visions.

Proteomics is progressing at an unprecedented pace, as can be exemplified by the progress in model organisms such as yeast, bacteria, and mammals. Proteomics research in plants, however, has not progressed at the same pace. Unscrambling of the genome sequences of the dicotyledoneous Arabidopsis thaliana (L.) and monocotyledoneous rice (Oryza sativa L.) plant species, respectively, has made them accessible reference organisms to study plant proteomics. Study of these two reference plants is expected to unravel the mystery of plant biology. Rice, a critically important food crop on the earth, has been termed a "cornerstone" and the "Rosetta stone" for functional genomics of cereal crops. Here, we look at the progress in unraveling rice proteomes and present the facts, challenges, and vision. The text is divided into two major parts: the first part presents the facts and the second part discusses the challenges and vision. The facts include the technology and its use in developing proteomes, which have been critically and constructively reviewed. The challenges and vision deal with the establishment of technologies to exhaustively investigate the protein components of a proteome, to generate high-resolution gel-based reference maps, and to give rice proteomics a functional dimension by studying PTMs and isolation of multiprotein complexes. Finally, we direct a vision on rice proteomics. This is our third review in series on rice proteomics, which aims to stimulate an objective discussion among rice researchers and to understand the necessity and impact of unraveling rice proteomes to their full potential.

Arabidopsis↗

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↗

PubMed2006

The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols.

A set of homozygous diploid deletion mutants of the yeast Saccharomyces cerevisiae was screened for the genes required for tolerance to aliphatic alcohols. The screen identified 137, 122 and 48 deletion mutants sensitive to ethanol, 1-propanol and 1-pentanol, respectively. A number of the genes required for ethanol tolerance were those also required for tolerance to other alcohols. Numerous mutants with defective genes encoding for vacuolar H+ -ATPase (V-ATPase) were cosensitive to these alcohols. A global screening approach of yeast deletion library mutants was useful in elucidating the mechanisms of alcohol tolerance based on different lipophilicities.

1-Propanol↗

PubMed2006

A knockout strain of CPR1 induced during fermentation of Saccharomyces cerevisiae KNU5377 is susceptible to various types of stress.

To investigate the tolerance factor of Saccharomyces cerevisiae KNU5377 against various types of environmental stress during fermentation, we identified the protein that is upregulated at high temperatures. The highly upregulated protein was high-score-matched as a cytoplasmic peptidyl-prolyl cis-trans isomerase, cyclophilin (Cpr1p), by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). We constructed a CPR1-deleted KNU5377 strain (KNU5377Y cpr1Delta) to determine the roles of the protein under fermentative or stress condition. The growth of the S. cerevisiae KNU5377Y cpr1Delta strain was completely inhibited under the following conditions: heat (40 degrees C), hydrogen peroxide (20-30 mM), menadione (0.3 mM), ethanol (16%), sulfuric acid (5 mm), and lactic acid (0.4-0.8%). However, the wild-type and cpr1Delta mutant of S. cerevisiae BY4741 as a positive control did not show differences in sensitivity to stress. It is interesting to note that the wild-type KNU5377Y and KNU5377Y cpr1Delta mutant showed high sensitivity against various stresses, particularly, acid stress such as in the presence of sulfuric and lactic acid. Although the alcohol fermentation rate of the KNU5377Y cpr1Delta mutant markedly decreased with an increase in temperature up to 40 degrees C, we observed no decrease in that of the wild-type strain under the same conditions. These results suggest that CPR1 contributes to the stress tolerance of KNU5377 against various types of environmental stress caused during fermentation, thus leading to the physiological role of maintaining an alcohol fermentation yield, even at high temperatures such as 40 degrees C.

Cell Culture Techniques↗

PubMed2005

Genome-wide expression analysis of yeast response during exposure to 4 degrees C.

Adaptation to temperature fluctuation is essential for the survival of all living organisms. Although extensive research has been done on heat and cold shock responses, there have been no reports on global responses to cold shock below 10 degrees C or near-freezing. We examined the genome-wide expression in Saccharomyces cerevisiae, following exposure to 4 degrees C. Hierarchical cluster analysis showed that the gene expression profile following 4 degrees C exposure from 6 to 48 h was different from that at continuous 4 degrees C culture. Under 4 degrees C exposure, the genes involved in trehalose and glycogen synthesis were induced, suggesting that biosynthesis and accumulation of those reserve carbohydrates might be necessary for cold tolerance and energy preservation. The observed increased expression of phospholipids, mannoproteins, and cold shock proteins (e.g., TIP1) is consistent with membrane maintenance and increased permeability of the cell wall at 4 degrees C. The induction of heat shock proteins and glutathione at 4 degrees C may be required for revitalization of enzyme activity, and for detoxification of active oxygen species, respectively. The genes with these functions may provide the ability of cold tolerance and adaptation to yeast cells.

Adaptation, Physiological↗

PubMed2005

The stress response against denatured proteins in the deletion of cytosolic chaperones SSA1/2 is different from heat-shock response in Saccharomyces cerevisiae.

BACKGROUND: A yeast strain lacking the two genes SSA1 and SSA2, which encode cytosolic molecular chaperones, acquires thermotolerance as well as the mild heat-shocked wild-type yeast strain. We investigated the genomic response at the level of mRNA expression to the deletion of SSA1/2 in comparison with the mild heat-shocked wild-type using cDNA microarray. RESULTS: Yeast cDNA microarray analysis revealed that genes involved in the stress response, including molecular chaperones, were up-regulated in a similar manner in both the ssa1/2 deletion mutant and the mild heat-shocked wild-type. Genes involved in protein synthesis were up-regulated in the ssa1/2 deletion mutant, but were markedly suppressed in the mild heat-shocked wild-type. The genes involved in ubiquitin-proteasome protein degradation were also up-regulated in the ssa1/2 deletion mutant, whereas the unfolded protein response (UPR) genes were highly expressed in the mild heat-shocked wild-type. RT-PCR confirmed that the genes regulating protein synthesis and cytosolic protein degradation were up-regulated in the ssa1/2 deletion mutant. At the translational level, more ubiquitinated proteins and proteasomes were detected in the ssa1/2 deletion mutant, than in the wild-type, confirming that ubiquitin-proteasome protein degradation was up-regulated by the deletion of SSA1/2. CONCLUSION: These results suggest that the mechanism for rescue of denatured proteins in the ssa1/2 deletion mutant is different from that in the mild heat-shocked wild-type: Activated protein synthesis in the ssa1/2 deletion mutant supplies a deficiency of proteins by their degradation, whereas mild heat-shock induces UPR.

Adenosine Triphosphatases↗

PubMed2005

Adaptation of Saccharomyces cerevisiae to high hydrostatic pressure causing growth inhibition.

Genome-wide mRNA expression profiles of Saccharomyces cerevisiae growing under hydrostatic pressure were characterized. We selected a hydrostatic pressure of 30 MPa at 25 degrees C because yeast cells were able to grow under these conditions, while cell size and complexity were increased after decompression. Functional characterization of pressure-induced genes suggests that genes involved in protein metabolism and membrane metabolism were induced. The response to 30 MPa was significantly different from that observed under lethal conditions because protein degradation was not activated under 30 MPa pressure. Strongly induced genes those that contribute to membrane metabolism and which are also induced by detergents, oils, and membrane stabilizers.

Adaptation, Physiological↗

PubMed2005

The rice (Oryza sativa) blast lesion mimic mutant, blm, may confer resistance to blast pathogens by triggering multiple defense-associated signaling pathways.

Here we characterized a rice (Oryza sativa L.) blast lesion mimic (blm) mutant, identified previously in an N-methyl-N-nitrosourea-mutagenized population of the cultivar Hwacheong (wild type). The rice blm displayed spontaneous necrotic lesion formation on the leaves during development under long-day condition and temperature shift from 28 to 24 degrees C in the absence of obvious stress/disease, and provided us with a highly reproducible and convenient experimental system in the growth chamber to study blm. The blm phenotype resembled to the cell death of hypersensitive reaction (HR), and subsequent, two-dimensional gel electrophoresis (2-DGE) revealed induction of many leaf proteins; prominent among them were the three pathogenesis-related (PR) marker proteins of class 5 (one spot) and 10 (two spots). Interestingly, the rice blm manifested HR against all races tested of the rice blast fungus (Magnaporthe grisea), providing high resistance in a non-race specific manner. It was also observed that blm was highly resistant to hydrogen peroxide treatment. Using 2-DGE immunoblotting, we identified the presence of 4 new spots cross-reacting with a superoxide dismutase (SOD) antibody, only in blm, suggesting the expression of potentially new SOD protein (isoforms) during lesion formation. In the leaves of blm, autofluorescent compounds accumulated in and around the site of lesion progression. Moreover, enhanced levels of two major rice phytoalexins, sakuranetin and momilactone A were also observed in the leaves of blm. These results indicate that blm confers broad-spectrum resistance to multiple pathogens, and so, it could be hypothesized that the BLM gene product may control the HR-like cell death and its associated multiple defense signaling pathways, as evidenced by induction of known hallmark features (proteins/metabolites) linked with the defense responses, in rice.

Antifungal Agents↗

PubMed2005

System, trends and perspectives of proteomics in dicot plants. Part III: Unraveling the proteomes influenced by the environment, and at the levels of function and genetic relationships.

This review is devoted to the proteomics studies in dicotyledoneous (dicot) plants, such as Arabidopsis, Medicago, potato, soybean, and tomato, under the influence of the environment and at the functional and genetic relationship levels, where the two core technologies, two-dimensional gel electrophoresis (2-DGE) and mass spectrometry (MS) have been instrumental in unraveling the proteomes affected therein. Abiotic and biotic stress responses, including the affect of allergens, the symbiotic interaction between the members of the Leguminoseae family and genera of nitrogen fixing bacteria, phosphoproteomics, and proteomics in revealing the genetic relationships between species and genera have been the subject of many proteomics studies, and these are discussed in this review. In all, these studies have complemented and extended the studies of developmental proteomics [G.K. Agrawal, M. Yonekura, Y. Iwahashi, H. Iwahashi, R. Rakwal, J. Chromatogr. B (2004)].

Cold Temperature↗