Biomedical subjects

Hitoshi Iwahashi

Publications and source records attributed to Hitoshi Iwahashi.

At least 19 recordsLinked to original sources

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↗

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↗

PubMed2003

Rice MAPKs.

Mitogen-activated protein kinase (MAPK) cascades are evolutionary conserved from unicellular to complex eukaryotic organisms, and constitute one of the major signalling pathways involved in regulating a wide range of cellular activities from growth and development to cell death. MAPKs of rice (Oryza sativa L.), the most important of all food crops and an established monocot plant research model, have seen considerable progress mainly on their identification and characterization during the past one year alone. These studies have provided new information on the response and regulation of rice MAPKs, in particular on their possible role/function in the rice self-defense pathways. It is believed that further work on MAPK cascades in rice will widen our understanding of the MAPK signalling pathways, and may lead to the establishment of a biological model on this critical early signalling event in monocots. In this review, we bring together all the recent developments in rice MAPKs and discuss their significance and future direction in light of the present data and the progress made in dicot model plants.

Enzyme Activation↗

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↗

PubMed2003

Screening of genes that respond to cryopreservation stress using yeast DNA microarray.

We studied the response of yeast cells after cryopreservation treatment using DNA microarray technology. Genes that contribute to "Cell rescue, defense and virulence," "energy," and "metabolism," were significantly induced. These genes were classified as encoding heat shock proteins, oxidative stress scavenger, and enzymes involved in glucose metabolism. The expression profile of mRNA after cryopreservation treatment was calculated to be closer to that following treatment with detergent or plant oils rather than by other stress factors such as heavy metals and agricultural chemicals. These results suggest that the cryopreservation treatment caused damage to the structure of the cell wall and cellular organelles. This was supported by the localization of the products of the induced genes at the cell wall and within cellular organelles.

Cell Wall↗

PubMed2003

Yeast gene expression during growth at low temperature.

Gene expression during growth at low temperature in the yeast Saccharomyces cerevisiae was investigated by means of DNA microarray analysis. A large number of genes showed an increase or decrease in expression at 4 degrees C relative to 25 degrees C. Although a temperature shift was not performed, differential expression of the cold shock genes TIP1, TIR1, TIR2, and NSR1 was observed. These genes may be necessary for growth at temperatures as low as 4 degrees C as well as for adapting to rapid drops in temperature. A new class of genes, many with unknown functions, was found to be induced during growth at low temperature. We propose to call these genes "low temperature growth genes."

Cold Temperature↗

PubMed2002

Effects of the pesticide thiuram: genome-wide screening of indicator genes by yeast DNA microarray.

Although there have been studies on the toxicity of the pesticide thiuram, the present study is the first one to attempt to integrate a whole genomic response using microarray technology. From the DNA microarray experiment it was found that exposure to thiuram led to alterations of gene expression in yeast cells and that many genes involved in detoxification and stress response were highly induced. The induced genes were classified according to the MIPS yeast database. The induction of genes concerned with folding and proteolysis reflects the protein denaturing and degradation effects of the thiuram treatment The induction of genes involved in redox and defense against reaction oxygen species also suggests that thiuram has other effects, such as oxidative stress. Genes classified for carbohydrate metabolism and energy were also highly induced, and these gene products may play the role of providing the energy for the detoxification mechanism. In addition, in view of the induction of some genes involved in DNA repair, thiuram potentially causes DNA damage. Therefore, as stated in previous reports, thiuram is a potential positive toxic chemical. On the other hand, YKL071W, YCR102C, YLR303W, and YLL057C were selected based on the result of a DNA microarray experiment and used for the promoter activity assay. Thiuram treatment affected the promoter of these genes, indicating that this technique could be used for the selection of biomarker candidates.

Biomarkers↗

PubMed2002

Octadecanoid signaling component "burst" in rice (Oryza sativa L.) seedling leaves upon wounding by cut and treatment with fungal elicitor chitosan.

Octadecanoid pathway components, 12-oxo-phytodieonic acid (OPDA) and jasmonic acid (JA), are key biologically active regulators of plant self-defense response(s). However, to date these compounds have been studied mostly in dicots, and used large (1-10 g fresh weight, FW) samples for quantification, even when examined in mature rice plants, which is a drawback considering their rapid responsiveness to stress. Focusing on rice--a monocot cereal crop research model--this work describes an efficient and simultaneous quantification of both OPDA and JA using a minimum amount of 200mg FW seedling leaf tissue upon wounding (by cut) and treatment with fungal elicitor, chitosan (CT) by high-pressure liquid chromatography-turboionspray tandem mass spectrometry. Transient OPDA/JA "burst" was consistently and reproducibly detected within 3 min in wounded and CT treated leaves. OPDA peaked dramatically around 5 min and returned to its basal level within 15 min, whereas JA induction upon wounding and CT treatment were in parallel to OPDA production, peaking at 30 and 60 min, respectively. Present results mark a major advance in our understanding of key inducible octadecanoid pathway components in rice, and strongly suggest a role for the octadecanoid pathway downstream of perception of at least these two fundamentally different extracellular stimuli.

Antigens, Fungal↗

PubMed2002

Isolation of novel rice (Oryza sativa L.) multiple stress responsive MAP kinase gene, OsMSRMK2, whose mRNA accumulates rapidly in response to environmental cues.

In search for components of MAPK (mitogen-activated protein kinase) cascades in rice (Oryza sativa L. cv. Nipponbare), we identified a single copy gene called OsMSRMK2 from jasmonic acid (JA) treated rice seedling leaf cDNA library. This gene has a conserved protein kinase domain, including a MAPK family signature, and encodes a 369 amino acid polypeptide with a predicted molecular mass of 42995.43 and a pI of 5.48. OsMSRMK2 did not show constitutive expression in leaves and was induced within 15 min in response to wounding by cut. Using in vitro system, we show that the expression of OsMSRMK2 mRNA was potently enhanced within 15 min by signalling molecules, protein phosphatase inhibitors, ultraviolet irradiation, fungal elicitor, heavy metals, high salt and sucrose, and drought. OsMSRMK2 expression was further modulated by co-application of JA, salicylic acid, and ethylene and required de novo synthesized protein factor(s) in its transient regulation. Moreover, high (37 degrees C) and low temperatures (12 degrees C) and environmental pollutants-ozone and sulfur dioxide-differentially regulate the OsMSRMK2 mRNA accumulation in leaves of intact plants. Present results demonstrating dramatic transcriptional and transient regulation of the OsMSRMK2 expression by diverse biotic/abiotic stresses, a first report for any rice (or plant) MAPK to date, suggest a role for OsMSRMK2 in rice defense/stress response pathways.

Amino Acid Sequence↗