Ken-ichi Yamazaki

The Arabidopsis transcriptional repressor ERF9 participates in resistance against necrotrophic fungi

Complex plant defenses that include the hypersensitive response (HR) are mediated by plant hormones, such as salicylic acid (SA), jasmonic acid (JA) and ethylene. We previously isolated the Arabidopsis DEAR1 (DREB AND EAR MOTIF PROTEIN 1) regulator and showed that its overexpression DEAR1 (DEAR1ox) resulted in a dwarf phenotype and lesion-like cell death, accompanied by elevated expression of PR (PATHOGENESIS-RELATED) genes. Here, we show that transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) has enhanced resistance to the necrotrophic fungus Botrytis cinerea (B. cinerea). This result indicates that DEAR1 represses negative regulators of plant defense responses, including transcriptional repressors belonging to the ERF (ETHYLEN RESPONSE FACTOR) family. Knockout mutants of ERF9 (erf9), which were down-regulated in DEAR1ox plants, showed transcriptional promotion of PDF1.2 (PATHOGEN-INDUCIBLE PLANT DEFENSIN) genes, which serve as positive markers for the ethylene/jasmonic acid (JA) signaling pathway and provide enhanced resistance to B. cinerea. Biochemical assays demonstrated that the ERF9 in capable of binding to the GCC box, a cis-element contained in the promoters of the PDF1.2 gene that possesses trans-repression activity. Moreover, infection with B. cinerea resulted in the promotion of the PDF1.2 expression, coinciding with suppression of the ERF9 gene under the control of the DEAR1 gene. These results indicate that the transcriptional repressor ERF9 participates in plant defense mechanisms against necrotic fungi mediated by the DEAR1-dependent ethylene/JA signaling pathway.

Differentially expressed genes associated with CIS-diamminedichloroplatinum (II) resistance in head and neck cancer using differential display and CDNA microarray

The mechanism by which cancer cells become resistant to cis‐Diamminedichloroplatinum (II) (cDDP) is not completely understood. To investigate the molecular markers involved in the cDDP resistance, we compared the gene expression profiles between a head and neck squamous cell carcinoma (HNSCC) line sensitive to cDDP and its cDDP‐resistant variant.

The analysis of an Arabidopsis triple knock-down mutant reveals functions for MBF1 genes under oxidative stress conditions.

Transcriptional co-activators of the multiprotein bridging factor 1 (MBF1) type belong to a small multigenic family that controls gene expression by connecting transcription factors and the basal transcription machinery. In this report, a triple knock-down mutant (abc-) for the Arabidopsis thaliana MBF1 genes AtMBF1a, AtMBF1b and AtMBF1c was generated. The phenotypic characterization using oxidative agents such as hydrogen peroxide and methyl viologen revealed that the abc- mutant was more sensitive to oxidative stress. The triple knock-down mutant, abc- was also sensitive to osmotic stress mediated by high concentrations of sorbitol. Furthermore, the abc- phenotype was partially or completely rescued by AtMBF1c cDNA over-expression (abc- +c) depending on physiological and developmental conditions. AtMBF1s regulate the expression of ABR1, which is a member of the ethylene-response factor family and acts as ABA repressor. Thus, we conclude that AtMBF1 gene family may function as a regulatory component of the cross-talk node between ethylene, ABA and stress signal pathways. Furthermore, higher levels of a HSP70 mRNA and an immunoreactive HSP70 protein were detected in the abc- mutant. The participation of MBF1c as a possible negative regulator of HSP genes was discussed.

SOLUTION STRUCTURE OF ALPHA -CONOTOXIN MI DETERMINED BY 1H-NMR SPECTROSCOPY AND MOLECULAR DYNAMICS SIMULATION WITH THE EXPLICIT SOLVENT WATER

Abstract The conformation of α-conotoxin MI, a potent antagonist of the nicotinic acetylcholine receptor, has been investigated in aqueous solution. Two-dimensional NMR experiments and simulated annealing calculations provide the overall topology of α-conotoxin MI; then molecular dynamics simulation with the explicit solvent water was followed in order to obtain a more reliable solution structure. The resulting conformation indicates the presence of a 310 helix and a type I β-turn for residues Pro6–Cys8 and Gly9–Try12, respectively, and shows a significant structural similarity to that of α-conotoxin GI, which has biological activity similar to that of MI. The present study provides a molecular basis for the α-conotoxin–receptor interaction.

TATA SEQUENCE REQUIREMENTS FOR THE INITIATION OF TRANSCRIPTION FOR AN RNA POLYMERASE II IN VITRO TRANSCRIPTION SYSTEM FROM NICOTIANA TABACUM

This is the first report to describe the detailed analysis of TATA sequence requirements for the initiation of transcription for RNA polymerase II in a plant in vitro transcription system. We examined the transcriptional activities associated with 24 promoters, including wild-type (TATATATA), and the 21 point and two multiple mutations in tobacco, Drosophila, and HeLa in vitro systems. Although the relative transcriptional activities varied among these promoters, all three systems gave similar results. These results suggest the functional conservation of TATA sequence requirements for in vitro transcription systems among plants, insects, and human

Arabidopsis MBF1s control leaf cell cycle and its expansion.

Multiprotein bridging factor 1 (MBF1) is known as a transcriptional co-activator that enhances transcription of its target genes by bridging between transcription factors and TATA-box-binding protein in eukaryotes. Arabidopsis thaliana has three MBF1 genes: AtMBF1a-AtMBF1c. However, details of the functions of AtMBF1 remain unclear. For this study, transgenic Arabidopsis overexpressing AtMBF1 fused to an active transcriptional repression domain (SRDX) was constructed. The chimeric protein putatively functions as a transcriptional co-repressor and as a suppressor of functions of endogenous AtMBF1 in transgenic plants. Transgenic Arabidopsis overexpressing AtMBF1-SRDX (AtMBF1-SRDX(OE)) showed an extremely small leaf phenotype under a continuous white light condition. Its leaf cells-especially those around vascular tissues, where strong expression of endogenous AtMBF1s is observed-were much smaller than those from the wild type (WT). In addition, a lower cell number was observed in leaves from AtMBF1-SRDX(OE) plants. Time course analysis of cell size revealed that cell expansion of leaves of AtMBF1-SRDX(OE) plants was dramatically suppressed during the late leaf developmental stage (cell expansion stage), when endogenous AtMBF1b is strongly expressed in the WT. The results show that ploidy levels of leaves from AtMBF1-SRDX(OE) plants were dramatically lower than those from the WT; moreover, expression levels of several negative regulators of endoreduplication were more elevated in AtMBF1s-SRDX(OE) plants than those in the WT. These observations suggest that AtMBF1-SRDX interacts with regulators of endoreduplication. Therefore, AtMBF1s are considered to affect not only leaf cell expansion but also regulation of the ploidy level in leaf cells during the leaf expansion stage.

Transcriptional coactivator MBF1s from Arabidopsis predominantly localize in nucleolus

Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator. It has been reported that MBF1 changed its subcellular localization from cytoplasm into nuclei with a transcriptional activator for activation of a target gene expression in animals. We found that Arabidopsis MBF1s (AtMBF1s) predominantly localize in nucleolus. We previously reported that plant MBF1s were rapidly induced by several stresses, whereas animal MBF1s were not induced. Therefore, we suggest that MBF1-function in plants is controlled on the level of transcriptional induction but not by nuclear translocation, dissimilar from the case of MBF1s from animals.

MBF1s regulate ABA-dependent germination of Arabidopsis seeds

Transcriptional co-activators of the multiprotein bridging factor 1 (MBF1) control gene expression by connecting transcription factors and the basal transcription machinery. In Arabidopsis thaliana functions of MBF1 genes have been related to stress tolerance and developmental alterations. Endogenous ABA plays a major role in the regulation of Arabidopsis seed dormancy and germination. Seed dormancy and ABA sensitivity are enhanced in ethylene insensitive mutants suggesting that ethylene signal transduction pathway is necessary to fully develop ABA-dependent germination. In this report we showed that a triple knock-down mutant for Arabidopsis MBF1 genes (abc-) has enhanced seed dormancy and displays hypersensitivity to exogenous ABA. In addition, higher ABA contents were detected in abc- seeds after imbibition. These evidences suggest a negative role of MBF1s genes in ABA-dependent inhibition of germination. The participation of MBF1s in ethylene signal transduction pathway is also discussed.

Biochemical and immunological characterization of rice homologues of the human immunodeficiency virus-1 Tat binding protein and subunit 4 of human 26S proteasome subunits

Previously, we isolated two cDNA clones, TBPOs-1 and TBPOs-2, encoding putative ATPases that are the rice homologues of human immunodeficiency virus-1 (HIV-1) Tat binding protein-1 and subunit 4 of human 26S proteasome. In order to determine the RNA-dependent ATPase activity of these putative proteins, the subclones from these cDNA clones were expressed in Escherichia coli as fusion proteins with maltose-binding protein. The recombinant proteins stimulated ATP hydrolysis in the presence of poly(U) and rice total RNA. In contrast, single- and double-stranded forms of HindIII-digested λ phage DNA are less effective at stimulating ATP hydrolysis. Western blot analysis using antisera against the TBPOs proteins showed a widespread appearance of these proteins in rice tissues and cultured cells. The TBPOs proteins were also found around the region where rice proteasomes would sediment. In addition, the TBPOs-1 protein bound to tobacco TATA-binding protein in vitro. Thus, we suggest that the TBPOs proteins are novel RNA-dependent ATPases characteristic of DEAD-box proteins and propose that the TPBOs proteins can exist in rice proteasomes. Further, the TBPOs-1 protein is thought to play a role in transcriptional events.

Toward an Implantable Axial Intra-Cardiac Blood Pump: The Valvo-Pump

We have developed the Valvo-Pump, an axial flow blood pump, which is implanted at the natural heart valve position. This device has several advantages; it fits well anatomically, its blood-contacting surface is small because the left or right ventricular cavity is used as the blood storage reservoir, and it is implanted as easily as an artificial heart valve replacement. The Valvo-Pump consists of an impeller and a motor, both of which are encased in a housing. An impeller with 5 vanes (22.0 mm in diameter) is connected to a samarium-cobalt-rare earth magnet direct current (DC) brushless motor (21.3 mm in diameter and 24.9-mm-long). Sealing is achieved by means of a ferro-fluidic seal. A pump flow of 16.11/min was obtained at a pump differential pressure of 7.7kPa (58mmHg), and a flow of 5.01/min at 18.4kPa (138mmHg) with a motor speed of 8500 revolutions per min (rpm). Sealing was kept perfect against a pressure of 29.3 kPa (220 mmHg) at 9000 rpm. The Valvo-Pump is a very promising and simple method of circulatory support.