Involvement of ethylene signaling in Azospirillum sp. B510-induced disease resistance in rice

A bacterial endophyte Azospirillum sp. B510 induces systemic disease resistance in the host without accompanying defense-related gene expression. To elucidate molecular mechanism of this induced systemic resistance (ISR), involvement of ethylene (ET) was examined using OsEIN2-knockdown mutant rice. Rice blast inoculation assay and gene expression analysis indicated that ET signaling is required for endophyte-mediated ISR in rice.

Abbreviations: ACC: 1-aminocyclopropane-1-carboxylic acid; EIN2: ethylene-insensitive protein 2; ET: ethylene; ISR: induced systemic resistance; JA: jasmonic acid; RNAi: RNA interference; SA: salicylic acid; SAR: systemic acquired resistance     

A fibroblast-derived tumor cytotoxic factor/F-TCF (hepatocyte growth factor/HGF) has multiple functions in vitro.

We previously demonstrated that a tumor cytotoxic factor(F-TCF) purified from the culture broth of human embryonic lung diploid fibroblast, IMR-90 cells was one of the human hepatocyte growth factors (hHGFs). In the present report, we demonstrate its biological functions. F-TCF showed moderate cytotoxicity on human tumor cell lines KB, BG-1, MCF-7 and Hs913 T, and strong cytotoxicity on mouse tumor cell lines Sarcoma 180, Meth A sarcoma and P388. On the contrary, F-TCF was a potent mitogen not only for adult rat hepatocytes, but also for human endothelial cells, HUVEC and human melanocytes. Moreover, F-TCF induced the differentiation of premyelocyte leukemia, HL-60 cells into morphologically granulocyte-like cells. These biological functions suggest that F-TCF is an effector molecule responsible for inflammation and repair in injured tissues including tumor and liver.     

Crystal structures of the state 1 conformations of the GTP-bound H-Ras protein and its oncogenic G12V and Q61L mutants

GTP-bound Ras adopts two interconverting conformations, "inactive" state 1 and "active" state 2. However, the tertiary structure of wild-type (WT) state 1 remains unsolved. Here we solve the state 1 crystal structures of H-Ras WT together with its oncogenic G12V and Q61L mutants. They assume open structures characterized by impaired interactions of both Thr-35 in switch I and Gly-60 in switch II with the γ-phosphate of GTP and possess two surface pockets of mutually different shapes unseen in state 2, a potential target for selective inhibitor development. Furthermore, they provide a structural basis for the low GTPase activity of state 1.     

Phenotype-based identification of mouse chromosome instability mutants

There is increasing evidence that defects in DNA double-strand-break (DSB) repair can cause chromosome instability, which may result in cancer. To identify novel DSB repair genes in mice, we performed a phenotype-driven mutagenesis screen for chromosome instability mutants using a flow cytometric peripheral blood micronucleus assay. Micronucleus levels were used as a quantitative indicator of chromosome damage in vivo. Among offspring derived from males mutagenized with the germline mutagen N-ethyl-N-nitrosourea (ENU), we identified a recessive mutation conferring elevated levels of spontaneous and radiation- or mitomycin C-induced micronuclei. This mutation, named chaos1 (chromosome aberration occurring spontaneously 1), was genetically mapped to a 1.3-Mb interval on chromosome 16 containing Polq, encoding DNA polymerase theta. We identified a nonconservative mutation in the ENU-derived allele, making it a strong candidate for chaos1. POLQ is homologous to Drosophila MUS308, which is essential for normal DNA interstrand crosslink repair and is unique in that it contains both a helicase and a DNA polymerase domain. While cancer susceptibility of chaos1 mutant mice is still under investigation, these data provide a practical paradigm for using a forward genetic approach to discover new potential cancer susceptibility genes using the surrogate biomarker of chromosome instability as a screen.     

Involvement of the smeAB multidrug efflux pump in resistance to plant antimicrobials and contribution to nodulation competitiveness in Sinorhizobium meliloti

The contributions of multicomponent-type multidrug efflux pumps to antimicrobial resistance and nodulation ability in Sinorhizobium meliloti were comprehensively analyzed. Computational searches identified genes in the S. meliloti strain 1021 genome encoding 1 pump from the ATP-binding cassette family, 3 pumps from the major facilitator superfamily, and 10 pumps from the resistance-nodulation-cell division family, and subsequently, these genes were deleted either individually or simultaneously. Antimicrobial susceptibility tests demonstrated that deletion of the smeAB pump genes resulted in increased susceptibility to a range of antibiotics, dyes, detergents, and plant-derived compounds and, further, that specific deletion of the smeCD or smeEF genes in a ΔsmeAB background caused a further increase in susceptibility to certain antibiotics. Competitive nodulation experiments revealed that the smeAB mutant was defective in competing with the wild-type strain for nodulation. The introduction of a plasmid carrying smeAB into the smeAB mutant restored antimicrobial resistance and nodulation competitiveness. These findings suggest that the SmeAB pump, which is a major multidrug efflux system of S. meliloti, plays an important role in nodulation competitiveness by mediating resistance toward antimicrobial compounds produced by the host plant.     

ATP9B, a P4-ATPase (a putative aminophospholipid translocase), localizes to the trans-Golgi network in a CDC50 protein-independent manner

Type IV P-type ATPases (P4-ATPases) are putative phospholipid flippases that translocate phospholipids from the exoplasmic (lumenal) to the cytoplasmic leaflet of lipid bilayers and are believed to function in complex with CDC50 proteins. In Saccharomyces cerevisiae, five P4-ATPases are localized to specific cellular compartments and are required for vesicle-mediated protein transport from these compartments, suggesting a role for phospholipid translocation in vesicular transport. The human genome encodes 14 P4-ATPases and three CDC50 proteins. However, the subcellular localization of human P4-ATPases and their interactions with CDC50 proteins are poorly understood. Here, we show that class 5 (ATP10A, ATP10B, and ATP10D) and class 6 (ATP11A, ATP11B, and ATP11C) P4-ATPases require CDC50 proteins, primarily CDC50A, for their exit from the endoplasmic reticulum (ER) and final subcellular localization. In contrast, class 2 P4-ATPases (ATP9A and ATP9B) are able to exit the ER in the absence of exogenous CDC50 expression: ATP9B, but not ATP11B, was able to exit the ER despite depletion of CDC50 proteins by RNAi. Although ATP9A and ATP9B show a high overall sequence similarity, ATP9A localizes to endosomes and the trans-Golgi network (TGN), whereas ATP9B localizes exclusively to the TGN. A chimeric ATP9 protein in which the N-terminal cytoplasmic region of ATP9A was replaced with the corresponding region of ATP9B was localized exclusively to the Golgi. These results indicate that ATP9B is able to exit the ER and localize to the TGN independently of CDC50 proteins and that this protein contains a Golgi localization signal in its N-terminal cytoplasmic region.     

Differential gene dosage effects of Ad4BP/SF-1 on target tissue development

Ad4BP/SF-1 (NR5A1) was identified as a key regulator of the hypothalamus-pituitary-gonadal and -adrenal axes. Loss-of-function studies revealed that Ad4BP/SF-1 is essential for the development of these tissues and spleen. Here, we generated transgenic mouse with BAC recombinants carrying a dual promoter and Tet-off system. These recombinants have a potential to express lacZ and Ad4BP/SF-1 in the tissues where endogenous Ad4BP/SF-1 is expressed. However, protein level of Ad4BP/SF-1 varied among the tissues of the transgenic mice and probably thereby the target tissues are affected differentially. The BAC-transgenic mice were applied to rescue Ad4BP/SF-1 KO mouse. Interestingly, the mice successfully rescued the gonad and spleen but failed to rescue the adrenal gland. This variation might be dependent on in part the protein expression levels among the tissues and in part on differential sensitivities to the gene dosage.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A previous paper has demonstrated that enhanced tumor-specific immunity could be induced by priming mice with Bacillus Calmette Guerin (BCG) and subsequently immunizing them with syngeneic tumor cells modified with BCG-cross-reactive muramyl dipeptide (MDP) hapten [15]. The present study establishes a tumorspecific immunotherapy protocol for a murine chronic leukemia based on the above T-T cell collaboration between antitumor effector T cells and anti-MDP hapten helper T cells induced by BCG priming. BALB/c mice which had been primed to BCG were injected intravenously (i.v.) with viable, syngeneic BCL1 leukemia cells. One week later, these mice were immunized intraperitoneally (i.p.) with unmodified or MDP hapten-modified, 10,000 R X-irradiated BCL1 cells, followed by 4 booster immunizations at 5-day intervals. The administration of unmodified BCL1 tumor cells into BCG-primed mice failed to prevent them from tumor death due to the persistent growth of preinjected BCL1 cells. In contrast, the immunization of BCG-primed, BCL1 leukemia-cell-bearing mice with MDP-modified BCL1 cells resulted in a high growth inhibition of leukemia cells and protection of these mice from death by leukemia. It was also revealed that potent tumorspecific, T-cell-mediated immunity was generated in mice which survived in this immunotherapy model. Thus, these results indicate that administration of MDP hapten-modified, syngeneic leukemia cells into leukemia-bearing mice which have been primed with BCG results in potent tumor-specific, T-cell-mediated immunity attributable to preventing the growth of disseminated leukemic cells.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science, and Culture, Japan Abbreviations used: TATA, tumor-associated transplantation antigens; MDP, muramyl dipeptide; MTP, muramyl tripeptide; BCG, Bacillus Calmette Guerin     

Characterization of glutamate decarboxylase from a high gamma-aminobutyric acid (GABA)-producer, Lactobacillus paracasei

Gamma-aminobutyric acid (GABA) has several physiological functions in humans. We have reported that Lactobacillus paracasei NFRI 7415 produces high levels of GABA. To gain insight into the higher GABA-producing ability of this strain, we analyzed glutamate decarboxylase (GAD), which catalyzes the decarboxylation of L-glutamate to GABA. The molecular weight of the purified GAD was estimated to be 57 kDa by SDS-PAGE and 110 kDa by gel filtration, suggesting that GAD forms the dimer under native conditions. GAD activity was optimal at pH 5.0 at 50 degrees C. The Km value for the catalysis of glutamate was 5.0 mM, and the maximum rate of catalysis was 7.5 micromol min(-1) mg(-1). The N-terminal amino acid sequence of GAD was determined, and the gene encoding GAD from genomic DNA was cloned. The findings suggest that the ability of Lb. paracasei to produce high levels of GABA results from two characteristics of GAD, viz., a low Km value and activity at low pH.