Eiichiro Ichiishi

Premature progression of anther early developmental programs accompanied by comprehensive alterations in transcription during high-temperature injury in barley plants

High-temperature stress causes abortive male reproductive development in many plant species. Here, we report a putative mechanism of high-temperature injury during anther early development in barley plants (Hordeum vulgare L). Under high-temperature conditions (30°C day/25°C night), cell-proliferation arrest, increased vacuolization, over-development of chloroplasts, and certain abnormalities of the mitochondria, nuclear membrane, and rough endoplasmic reticulum (RER) were observed in developing anther cells, but not in developing ovule cells. Moreover, premature degradation of tapetum cells and premature progression to meiotic prophase in pollen mother cells (PMCs) were also observed. To monitor transcriptional alterations during high-temperature injury, we performed DNA microarray analysis using the 22K Barley1 GeneChip. Expression profiles were captured at four time points during the early development of panicles, and during vegetative growth of seedlings as a control, with or without high-temperature treatment. Abiotic or biotic stress related genes were equally or more dominantly up-regulated in the seedlings exposed to high temperatures compared with the panicles. In contrast, certain genes associated with histones, DNA replication initiation, mitochondria, and ribosomes were specifically repressed in the exposed panicles. In situ hybridization studies indicated that repression locally occurred on the developing anther cells exposed to high temperatures. Microarray analysis also indicated that a series of genes, including a meiosis-specific gene Asy1 and anther-specific lipid transfer protein genes, was prematurely up-regulated at an earlier stage under high-temperature conditions. Real-time quantitative RT-PCR analyses well confirmed the expression differences of certain key genes predicted by the DNA microarrays. These results suggest that high-temperature causes premature progression of anther early development program and fate, such as progression to meiosis of PMCs, cell-proliferation arrest and degradation in anther wall cells, accompanied by comprehensive alterations in transcription.

The effect of high strength static magnetic fields and ionizing radiation on gene expression and DNA damage in Caenorhabditis elegans.

Magnetic resonance imaging with high static magnetic fields (SMFs) has become widely used for medical imaging purposes because SMFs cause fewer genotoxic side effects than ionizing radiation (IR). However, the effect of exposure to high SMFs on global transcription is little understood. We demonstrate that genes involved in motor activity, actin binding, cell adhesion, and cuticles are transiently and specifically induced following exposure to 3 or 5 T SMF in the experimental model metazoan Caenorhabditis elegans. In addition, transient induction of hsp12 family genes was observed after SMF exposure. The small-heat shock protein gene hsp16 was also induced but to a much lesser extent, and the LacZ-stained population of hsp-16.1::lacZ transgenic worms did not significantly increase after exposure to SMFs with or without a second stressor, mild heat shock. Several genes encoding apoptotic cell-death activators and secreted surface proteins were upregulated after IR, but were not induced by SMFs. Real-time quantitative RT-PCR analyses for 12 of these genes confirmed these expression differences between worms exposed to SMFs and IR. In contrast to IR, exposure to high SMFs did not induce DNA double-strand breaks or germline cell apoptosis during meiosis. These results suggest that the response of C. elegans to high SMFs is unique and capable of adjustment during long exposure, and that this treatment may be less hazardous than other therapeutic tools.

Myristic Acid, A Side Chain of Phorbol Myristate Acetate (PMA), Can Activate Human Polymorphonuclear Leukocytes to Produce Oxygen Radicals More Potently than PMA.

Myristic acid (MyA), which is a saturated fatty acid (C14:0) and a side chain of phorbol 12-myristate 13-acetate (PMA), was examined if MyA stimulates human polymorphonuclear leukocytes (PMNs) to release oxygen radicals comparable to PMA by applying electron paramagnetic resonance (EPR)-spin-trapping method. When MyA was added to isolated human PMNs, spin adducts of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH and DMPO-OOH were time-dependently observed. The amounts of these spin adducts were larger than those of PMNs stimulated by PMA. These results clearly show that MyA is more potent agent to prime human PMNs than PMA, in a point of view of not only O2·− but also ·OH production. This fact calls attention that too much intake of MyA that is known to be contained vegetable oils can lead to crippling effect through uncontrolled production of reactive oxygen species.

Case study: Molecular dynamics study on the ligand recognition by tandem SH3 domains of p47phox, regulating NADPH oxidase activity

The phagocyte NADPH oxidase complex plays a crucial role in host defense against microbial infection through the production of superoxides. Chronic granulomatous disease (CGD) is an inherited immune deficiency caused by the absence of certain components of the NADPH oxidase. Key to the activation of the NADPH oxidase is the cytoplasmic subunit p47phox, which includes the tandem SH3 domains (N-SH3 and C-SH3). In active phagocytes, p47phox forms a stable complex with the cytoplasmic region of membrane subunit p22phox that forms a left-handed polyproline type-II (PPII) helix conformation. In this report, we have analyzed the conformational changes of p47phox-p22phox complexes of wild-type and three mutants, which have been detected in CGD patients, using molecular dynamics simulations. We have found that in the wild-type, two basal planes of PPII prism in cytoplasmic region of p22phox interacted with N-SH3 and C-SH3. In contrast, in the modeled mutants, the residue at the ape of PPII helix, which interacts simultaneously with both of the tandem SH3 domains in the wild-type, moved toward C-SH3. Furthermore, interaction energies of the cytoplasmic region of p22phox with C-SH3 tend to decrease in these mutants. All these findings led us to conclude that interactions between N-SH3 of p47phox and PPII helix, which is formed by cytoplasmic region of p22phox, may play a significant role in the activation of the NADPH oxidase.

Molecular evaluation using in silico protein interaction profiles

MOTIVATION To find a correlation between the activities and structures of molecules is one of the most important subjects for molecular evaluation study. Traditional quantitative structure-activity relationship (QSAR) methodologies represent those attempts using physicochemical descriptors. Creating a new molecular description factor based on the results of a computational docking study will add new dimensions to molecular evaluation. RESULTS We propose a new molecular description factor analysis system called the Comparative Molecular Interaction Profile Analysis (CoMIPA) system in which the AutoDock program is used for docking evaluation of small molecule compound-protein complexes. Interaction energies are calculated, and the data sets obtained are called interaction profiles (IPFs). Using the IPF as a scoring indicator, the system could be a powerful tool to cluster the interacting properties between small molecules and bio macromolecules such as ligand-receptor bindings. Further development of the system will enable us to predict the adverse effects of a drug candidate.

A graph theoretical approach for assessing bio-macromolecular complex structural stability

Fast and proper assessment of bio macro-molecular complex structural rigidity as a measure of structural stability can be useful in systematic studies to predict molecular function, and can also enable the design of rapid scoring functions to rank automatically generated bio-molecular complexes. Based on the graph theoretical approach of Jacobs et al. [Jacobs DJ, Rader AJ, Kuhn LA, Thorpe MF (2001) Protein flexibility predictions using graph theory. Proteins: Struct Funct Genet 44:150–165] for expressing molecular flexibility, we propose a new scheme to analyze the structural stability of bio-molecular complexes. This analysis is performed in terms of the identification in interacting subunits of clusters of flappy amino acids (those constituting regions of potential internal motion) that undergo an increase in rigidity at complex formation. Gains in structural rigidity of the interacting subunits upon bio-molecular complex formation can be evaluated by expansion of the network of intra-molecular inter-atomic interactions to include inter-molecular inter-atomic interaction terms. We propose two indices for quantifying this change: one local, which can express localized (at the amino acid level) structural rigidity, the other global to express overall structural stability for the complex. The new system is validated with a series of protein complex structures reported in the protein data bank. Finally, the indices are used as scoring coefficients to rank automatically generated protein complex decoys.

Effect of +36T > C in intron 1 on the glutamine: fructose-6-phosphate amidotransferase 1 gene and its contribution to type 2 diabetes in different populations

AbstractGlutamine: fructose-6-phosphate amidotransferase 1 (GFPT1) acts as a rate-limiting enzyme in the hexosamine biosynthetic pathway, which is an alternative branch of glucose metabolism. To evaluate GFPT1 as a susceptibility gene to type 2 diabetes, we surveyed the polymorphisms related with the gene function of GFPT1 and assessed its contribution to type 2 diabetes with a case-control association study. Screening of the 5′-flanking and all coding regions of GFPT1 revealed eight polymorphisms, one in the 5′-flanking region, one synonymous polymorphism in exon 8, five in introns and one in 3′-UTR, but no mis-sense or non-sense polymorphism. With in silico simulation, a putative promoter region was apparently predicted between 1 kb upstream and 1 kb downstream of the start codon. In this region, +36T > C polymorphism was located on the GC box sequence in intron 1, and its functional effect on promoter activity was confirmed by luciferase reporter assay, introducing a new functional polymorphism of the GFPT1 gene. To examine its association with type 2 diabetes, we analyzed 2,763 Japanese (1,461 controls and 1,302 cases) and 330 Caucasians (190 controls and 140 cases). One possible association of +36T > C was observed in Caucasians, but no association of polymorphisms including +36T > C in intron 1 or haplotypes was observed in Japanese. Although we could not completely rule out a contribution to specific sub-groups or other populations, genetic variation of GFPT1 is unlikely to have a major role in the susceptibility to type 2 diabetes in Japanese.

Lack of association of genetic variation in chromosome region 15q14-22.1 with type 2 diabetes in a Japanese population.

BackgroundChromosome 15q14-22.1 has been linked to type 2 diabetes (T2D) and its related traits in Japanese and other populations. The presence of T2D disease susceptibility variant(s) was assessed in the 21.8 Mb region between D15S118 and D15S117 in a Japanese population using a region-wide case-control association test.MethodsA two-stage association test was performed using Japanese subjects: The discovery panel (Stage 1) used 372 cases and 360 controls, while an independent replication panel (Stage 2) used 532 cases and 530 controls. A total of 1,317 evenly-spaced, common SNP markers with minor allele frequencies > 0.10 were typed for each stage. Captured genetic variation was examined in HapMap JPT SNPs, and a haplotype-based association test was performed.ResultsSNP2140 (rs2412747) (C/T) in intron 33 of the ubiquitin protein ligase E3 component n-recognin 1 (UBR1) gene was selected as a landmark SNP based on repeated significant associations in Stage 1 and Stage 2. However, the marginal p value (p = 0.0043 in the allelic test, OR = 1.26, 95% CI = 1.07–1.48 for combined samples) was weak in a single locus or haplotype-based association test. We failed to find any significant SNPs after correcting for multiple testing.ConclusionThe two-stage association test did not reveal a strong association between T2D and any common variants on chromosome 15q14-22.1 in 1,794 Japanese subjects. A further association test with a larger sample size and denser SNP markers is required to confirm these observations.

A Flexibility Index for Analysis of Bio-molecular Complexes

Molecular flexibility change at bio macro-molecular complex formation is an important factor in molecular recognition and function analysis. We propose an index to compute flexibility loss/gain at complex formation/separation. The algorithm performs a rigidity and/or flexibility evaluation of every node (atom) on the network constituted by all the intra and inter-molecular inter-atomic interactions. Comparison of flexible or rigid molecular regions or domains within the complex with those in the respective isolated monomers leads to the index, quantifying the loss (or gain) in the number of degrees of freedom at complex formation. The index can also be applied to ranking decoys output by docking algorithms.

An Evolving Automaton for RNA Secondary Structure Prediction

Conventional methods for RNA 2D structure prediction search for minimal free energy structures. RNAs, however, RNAs do not always adopt global minimum structures. Rather, their structure is the result of the folding pathway followed by the structure in nature, which adopts sub-optimal folds occurring along the pathway. Our algorithm consists of an automaton that generates RNA structures by searching for optimal folding pathways. The automaton is endowed of operations to travel throughout the hyperspace of conformers embedded in a base pairing matrix. Using genetic programming it evolves optimizing its ability to find optimal pathways and finally 2D structures. Comparing the evolving automaton with conventional methods shows its potential.