Biomedical subjects

Hitoshi Iwahashi

Publications and source records attributed to Hitoshi Iwahashi.

At least 37 records · Page 2Linked to original sources

PubMed2005

System, trends and perspectives of proteomics in dicot plants Part II: Proteomes of the complex developmental stages.

This review is devoted to the proteomes of the complex developmental stages of dicotyledoneous (dicot) plant materials. The two core technologies, two-dimensional gel electrophoresis (2-DGE) and mass spectrometry (MS), independently or in combination with each other, are propelling dicot plant proteomics to new discoveries and functions, with the establishment of tissue-specific and organelle proteomes, mostly in Arabidopsis thaliana and Medicago truncatula, revealing their complexity and specificity. These experimental proteomes have provided a good start towards the establishment of high-density 2-DGE reference maps and peptide mass fingerprint databases, for not only the model dicot plants, A. thaliana and M. truncatula, but also other important dicot plants, which will serve as a basis for proteomes of many other dicot plants and plant materials.

Arabidopsis↗

PubMed2005

System, trends and perspectives of proteomics in dicot plants Part I: Technologies in proteome establishment.

The first 3 years of the 21st century have seen the impact of plant proteomics on functional genomics that has enhanced our understanding, not only on the plant genome(s), but also more importantly, on the functional aspect of proteins. This is mainly due to availability of the complete genome sequence of the Arabidopsis thaliana-a dicotyledoneous (dicot) model plant-and technological advancements in proteomics. Proteomic analyses of a variety of dicot plants, including both Arabidopsis and the model legume species, barrel medic (Medicago truncatula), have greatly helped in an efficient separation, identification and cataloguing of a large number of proteins, and thereby defining their proteomes. Therefore, we have composed an inclusive review on dicot plant materials, as of February 2004, that provides system, trends and perspectives of proteomics in growth and development and the environment. The review is summarized and discussed as three individual, but interlinked, entities: Part I, technologies in proteome establishment (this review), Part II, proteomes of the complex developmental stages [G.K. Agrawal, M. Yonekura, Y. Iwahashi, H. Iwahashi, R. Rakwal, J. Chromatogr. B (2004)], and Part III, unraveling the proteomes influenced by the environment, and at the levels of function and genetic relationships [G.K. Agrawal, M. Yonekura, Y. Iwahashi, H. Iwahashi, R. Rakwal, J. Chromatogr. B (2004)]. This review deals with the diverse proteomic technologies being used in proteome development of different dicot plants.

Arabidopsis↗

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↗

PubMed2005

Effects of iodine on global gene expression in Saccharomyces cerevisiae.

It is well documented that iodine kills microorganisms with a broad spectrum, but a systematic study of its mechanism of action has not yet been reported. Here we found the action of iodine on gene expression level, using the yeast Saccharomyces cerevisiae with a DNA microarray. It was found that, like antimicrobial activity, iodine causes an immediate and dose-dependent (0.5 mM, 0.75 mM and 1 mM) transcriptional alteration in yeast cells. The effects of iodine continued after the first immediate response. Genes for c-compound and carbohydrate metabolism, for energy, and for cell rescue were continuously up-regulated. On the other hand, genes related to protein fate were induced especially at 0.5 h. The gene expression profile at 0.5 h was significantly different from that of a longer iodine exposed condition. The main reaction at 0.5 h after iodine addition might be due to oxidative toxicity, and the profile at 0.5 h was similar to that of an agricultural bactericide.

Carbohydrate Metabolism↗

PubMed2004

Response of Saccharomyces cerevisiae to a monoterpene: evaluation of antifungal potential by DNA microarray analysis.

Plant-derived essential oils with monoterpenoids have been used as antifungal drugs since ancient times, but the mode of action of these natural hydrocarbons at the molecular level is not understood. In order to understand the mechanisms of toxicity of alpha-terpinene (a cyclic monoterpene), a culture of Saccharomyces cerevisiae was exposed to 0.02% alpha-terpinene for 2 h and transcript profiles were obtained using yeast DNA arrays. These profiles, when compared with transcript profiles of untreated cultures, revealed that the expression of 793 genes was affected. For 435 genes, mRNA levels in treated cells compared with control cells differed by more than two-fold, whereas for 358 genes, it was <0.5-fold. Northern blots were performed for selected genes to verify the microarray results. Functional analysis of the up-regulated genes indicates that, similar to commonly used antifungal drugs, alpha-terpinene exposure affected genes involved in ergosterol biosynthesis and sterol uptake. In addition, transcriptional induction of genes related to lipid metabolism, cell wall structure and function, detoxification and cellular transport was observed in response to terpinene toxicity. Notably, the functions of 192 up-regulated genes are still unknown, but their characterization will probably shed light on the mechanisms of drug resistance and sensitivity. Taken together, this study showed that alpha-terpinene has strong antifungal activities and its modes of action resemble those of presently used antifungal drugs.

Antifungal Agents↗

PubMed2004

Inverse correlation between jasmonic acid and salicylic acid during early wound response in rice.

This study presents a kinetic analysis of the response to wounding in rice plants. In particular, jasmonic acid, salicylic acid, and lipoxygenase activity were measured in leaves of wounded rice plants during the early tillering phase. The results show that endogenous jasmonic acid transiently increases to a maximum 30 min after wounding (jasmonic acid burst) and lipoxygenase activity increases after the jasmonic acid burst, but not after the second smaller peak of endogenous jasmonic acid 23 h after wounding. In contrast, endogenous salicylic acid decreases during the jasmonic acid burst, such that the kinetic profiles of jasmonic acid and salicylic acid are inversely correlated during the early response to wounding. It is proposed here that the increase in endogenous jasmonic acid and the decrease in endogenous salicylic acid may contribute for establishing the efficient negative cross-talk between jasmonic acid and salicylic acid signaling pathways during the early response to wounding in rice.

Cyclopentanes↗

PubMed2004

Rice octadecanoid pathway.

Plant jasmonic acid (JA) and structurally similar animal prostaglandins play pivotal roles in regulating cellular responses against environmental cues, including the innate immune response(s). In plants, JA and its immediate precursor 12-oxo-phytodienoic acid (OPDA) are synthesized by the octadecanoid pathway, which employs at least five enzymes (lipase, lipoxygenase, allene oxide synthase and cyclase, and OPDA reductase), in addition to the enzymes involved in the beta-oxidation steps. Genetic, molecular, and biochemical analyses have led to the identification of almost all the genes of the octadecanoid pathway in Arabidopsis--a model dicotyledonous plant. In this regard, rice (Oryza sativa L.)--an important socio-economic monocotyledonous model research plant--remains poorly characterized. Until now, no gene has been specifically associated with this pathway. It is therefore of utmost importance to identify, characterize, and assign the pathway specific genes in rice. In this review, we have surveyed the rice genome, extracted a large number of putative genes of the octadecanoid pathway, and discussed their relationship with the known pathway genes from other plant species. Moreover, the achievements made so far on the rice octadecanoid pathway have also been summarized to reflect the contribution of rice towards extending our knowledge on this critical pathway in plants.

Animals↗

PubMed2004

A new approach to species determination for yeast strains: DNA microarray-based comparative genomic hybridization using a yeast DNA microarray with 6000 genes.

DNA-DNA hybridization is known as the superior method in the elucidation of relationships between closely related taxa, such as species and strain. For species determination we propose a new DNA-DNA hybridization method: the DNA microarray-based comparative genomic hybridization (CGH) method, using a yeast DNA microarray with approximately 6000 genes. The genome from a yeast strain as a sample strain (Sample) was labelled with Cy3-dye and hybridized to a single DNA microarray, together with the Cy5-labelled genome of S. cerevisiae S288C as a reference strain (Reference). The log2 ratio values [log2[Cy3(Sample)/Cy5(Reference)]: Ratio] of signal intensities of all the gene spots were estimated and divided into the following groups: Ratio < or = -1; -1 < Ratio < 1; 1 < or = Ratio. The hybridization profiles of the genomes of type strains belonging to the genus Saccharomyces were significantly different from that of S. cerevisiae S288C. The Ratio-based grouping allowed us to discriminate between some species from S. cerevisiae more clearly. Furthermore, cluster analysis discriminated between closely related species and strains. Using this method, we were able to not only perform species determination but also to obtain information on alternation in gene copy number of such gene amplifications and deletions with single-gene resolution. These observations indicated that DNA microarray-based CGH is a powerful system for species determination and comparative genome analysis.

Cluster Analysis↗

PubMed2004

Toxicity of anionic detergents determined by Saccharomyces cerevisiae microarray analysis.

Sodium n-dodecyl benzene sulfonate (LAS) and sodium dodecyl sulfate (SDS) are popular anionic detergents (surfactants) that are used worldwide and the toxicities of these chemicals have been characterized. We applied these chemicals in a DNA microarray bioassay and determined that the microarray data reflects previous findings and also provides some new information about anionic detergent toxicity. The mRNA expression profiles suggest that LAS and SDS cause damage to membranes and alterations in carbon metabolism, and induce the oxidative stress response. We also found that LAS and SDS induce the pleiotropic drug-resistance network, and that LAS and SDS may be pumped out of yeast cells by this network. Hierarchical clustering of the expression profiles showed that LAS and SDS cause similar features of toxicity and that the toxicity is similar to that of capsaicin but different from that of cadmium and mercury.

Benzenesulfonates↗

PubMed2004

Multiple reporter gene assays for the assessment and estimation of chemical toxicity.

To detect chemical toxicity, we are making new bioassay systems that use promoters selected from yeast DNA microarray experiments. We performed multiple reporter gene assays using the promoters of these genes; the promoter regions were inserted upstream of green fluorescence protein (GFP). In this report, six genes (HSP26, MET17, YLL057C, FIT2, CUP1 and OYE3) were selected and assays were carried out for 55 chemicals. The promoters of these genes showed different responses to chemicals within 4 h. This result indicates that this technique enables us to predict the toxicity of chemicals in the environment and to understand toxicities of newly synthesized chemicals.

Biological Assay↗

PubMed2004

Genomic profile of roundup treatment of yeast using DNA microarray analysis.

The herbicide Roundup, which contains glyphosate as the active ingredient, was first introduced in 1974 and has enjoyed widespread use in Japan and elsewhere in the world. Roundup-induced reactions occurring in the yeast Saccharomyces cerevisiae may have a predictive value for understanding responses in higher eukaryotes, and we applied yeast DNA microarray analysis for this purpose. Functional characterization of up-regulated open reading frames (ORFs) following Roundup treatment suggests that Roundup affects membrane structures and cellular organelles. Expression profiles induced by treatments with detergents, oils and hydrostatic pressure were similar to those following Roundup treatment based on cluster analysis. Glyphosate alone was not found to inhibit yeast growth at the concentration contained in the Roundup treatment used for microarray analysis. The toxicity of Roundup appeared to be due to detergent in the product.

DNA Damage↗

PubMed2004

Construction of a data dependent analysis (DDA) yeast cDNA microarray experiment for use in toxicogenomics.

DNA microarrays are becoming increasingly popular in toxicogenomic studies. It is common knowledge that multiple repeats of microarray experiments have to be performed with multiple samples to get reliable data, due to experimental variations (both technical and biological) contained in microarray experiments. However, the use of multiple samples and replicate experiments is not practical in the field of environmental toxicology, since environmental samples are limited and microarray experiments are expensive. Thus it is desirable to obtain reliable data from a minimum number of repeats of microarray experiments. To further establish and extend gene expression profiling for toxicogenomics using microarrays, it is necessary to establish an analytical method of microarrays for toxicogenomics. In this study, we attempted to construct a "Data Dependent Analysis (DDA)" yeast cDNA microarray experiment from 100 microarray datasets in order to get reliable data from one microarray experiment.

Carbocyanines↗

PubMed2003

Importance of ascorbate peroxidases OsAPX1 and OsAPX2 in the rice pathogen response pathways and growth and reproduction revealed by their transcriptional profiling.

We have identified and characterized two rice ascorbate peroxidase (OsAPX1 and OsAPX2) genes against diverse signals known to involve in defense/stress responses, using "2-week-old rice seedling in vitro model system". Oryza sativa ascorbate peroxidase 1 and 2 (OsAPX1/2) share 83% similarity to each other at amino acid level and belong to a small gene family in the rice genome. OsAPX1/2 show a weak constitutive expression in leaves. Their transcripts were up-regulated upon wounding (by cut), and diverse signals such as salicylic acid (SA), ethylene (using the ethylene generator, ethephon), abscisic acid (ABA), hydrogen peroxide, copper sulfate, protein phosphatase (PP) inhibitors, cantharidin (CN), endothall (EN) and okadaic acid (OA), and blast pathogen (Magnaporthe grisea) attack, but surprisingly not by jasmonic acid (JA). Interestingly, the OsAPX2 inductions were more potent and rapid than OsAPX1, whose expression increased gradually with time. These inductions were influenced by light signal(s), and showed requirement for certain de novo synthesized protein factor(s). Intriguingly, OsAPX1/2 mRNAs expression manifested a clear rhythmicity under light/dark cycle. Differential regulation of the OsAPX1/2 expressions by diverse environmental cues, including pathogen, and their developmental regulation in both young and reproductive stages strongly suggests their role in stress-signaling pathways and development in rice.

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

Small GTPase 'Rop': molecular switch for plant defense responses.

The conserved Rho family of GTPases (Rho, Rac, and Cdc42) in fungi and mammals has emerged as a key regulator of diverse cellular activities, such as cytoskeletal rearrangements, programmed cell death, stress-induced signaling, and cell growth and differentiation. In plants, a unique class of Rho-like proteins, most closely related to mammalian Rac, has only been found and termed 'Rop' (Rho-related GTPase from plant [Li et al. (1998) Plant Physiol. 118, 407-417; Yang (2002) Plant Cell 14, S375-S388]). ROPs have been implicated in regulating various plant cellular responses including defense against pathogens. It has been shown that ROPs, like mammalian Rac, trigger hydrogen peroxide production and hence the 'oxidative burst', a crucial component associated with the cell death, most likely via activation of nicotinamide adenine dinucleotide phosphate oxidase in both monocotyledonous and dicotyledonous species. Recent studies have established that ROPs also function as a molecular switch for defense signaling pathway(s) linked with disease resistance. As discerning the defense pathway remains one of the priority research areas in the field of plant biology, this review is therefore particularly focused on recent progresses that have been made towards understanding the plant defense responses mediated by ROPs.

Amino Acid Sequence↗

PubMed2003

Correlation of the structures of agricultural fungicides to gene expression in Saccharomyces cerevisiae upon exposure to toxic doses.

Correlations between the chemical structures of agricultural fungicides and mRNA expression levels following exposure of Saccharomyces cerevisiae to toxic doses of thiuram, zineb, maneb, TPN, and PCP were examined. Structurally, thiuram, zineb, and maneb are dithiocarbamate fungicides, whereas TPN and PCP are not. To characterize chemical toxicity, genes expression was classified according to the functional groups used by the MIPS database. However, no correlations between the classification scheme and chemical structures were found. Hierarchical clustering of gene expression profiles was performed to characterize the effects of the five chemicals. According to this analysis the similarity of gene expression profiles depended on the similarity of chemical structures. These results suggest that DNA microarray technology has potential for predicting the major chemicals which will cause environmental toxicity and will provide information on new biomonitoring methods.

Environmental Pollutants↗

PubMed2003

Dimethyl sulfoxide exposure facilitates phospholipid biosynthesis and cellular membrane proliferation in yeast cells.

Me2SO is a polar solvent that is widely used in biochemistry, pharmacology, and industry. Although there are several reports in the literature concerning the biological effects of Me2SO, the total cellular response remains unclear. In this paper, DNA microarray technology combined with the hierarchical clustering bioinformatics tool was used to assess the effects of Me2SO on yeast cells. We found that yeast exposed to Me2SO increased phospholipid biosynthesis through up-regulated gene expression. It was confirmed by Northern blotting that the level of INO1 and OPI3 gene transcripts, encoding key enzymes in phospholipid biosynthesis, were significantly elevated following treatment with Me2SO. Furthermore, the phospholipid content of the cells increased during exposure to Me2SO as shown by conspicuous incorporation of a lipophilic fluorescent dye (3,3'-dihexyloxacarbocyanine iodide) into the cell membranes. From these results we propose that Me2SO treatment induces membrane proliferation in yeast cells to alleviate the adverse affects of this chemical on membrane integrity.

Blotting, Northern↗

PubMed2003

Piezophysiology of genome wide gene expression levels in the yeast Saccharomyces cerevisiae.

Hydrostatic pressure is one of the physical factors affecting cellular physiology. Hydrostatic pressure of a few hundred MPa decreases the viability of yeast cells, and pressure of a few tens MPa decreases the growth rate. To understand the effect of hydrostatic pressure, we employed yeast DNA microarrays and analyzed genome-wide gene-expression levels after the pressure treatment with 180 MPa (immediate) at 4 degrees C and recovery incubation for 1 h and 40 MPa (16 h) at 4 degrees C and recovery incubation for 1 h. The transcription of genes involved in energy metabolism, cell defense, and protein metabolism was significantly induced by the pressure treatment. Genome-wide expression profiles suggested that high pressure caused damage to cellular organelles, since the induced gene products were localized in the membrane structure and/or cellular organelles. Hierarchical clustering analysis suggested that the damage caused by the pressure was similar to that caused by detergents, oils, and freezing/thawing. We also estimated the contribution of induced genes to barotolerance using some strains that have the deletion in the corresponding genes.

Animals↗