Expression Patterns and Potential Biological Roles of Dip2a

Disconnected (disco)-interacting protein 2 homolog A is a member of the DIP2 protein family encoded by Dip2a gene. Dip2a expression pattern has never been systematically studied. Functions of Dip2a in embryonic development and adult are not known. To investigate Dip2a gene expression and function in embryo and adult, a Dip2a-LacZ mouse model was generated by insertion of β-Gal cDNA after Dip2a promoter using CRISPR/Cas9 technology. Dip2a-LacZ mouse was designed to be a lacZ reporter mouse as well as a Dip2a knockout mouse. Heterozygous mice were used to study endogenous Dip2a expression and homozygotes to study DIP2A-associated structure and function. LacZ staining indicated that Dip2a is broadly expressed in neuronal, reproductive and vascular tissues, as well as in heart, kidney, liver and lung. Results demonstrate that Dip2a is expressed in ectoderm-derived tissues in developing embryos. Adult tissues showed rich staining in neurons, mesenchymal, endothelial, smooth muscle cells and cardiomyocytes by cell types. The expression pattern highly overlaps with FSTL1 and supports previous report that DIP2A to be potential receptor of FSTL1 and its protective roles of cardiomyocytes. Broad and intense embryonic and adult expression of Dip2a has implied their multiple structural and physiological roles.     

Germination stimulant from root exudates of Vigna unguiculata

Root exudate of Vigna unguiculata was extracted from a soil system consisting of charcoal and vermiculite. Germination stimulating activity for Striga gesnerioides was found in extracts of the soil system, and an active compound was isolated. The chemical structure of the active ingredient was determined to be (+)-4-O-acetylorobanchol, based on analysis of the spectral data of 1-D and 2-D NMR together with nuclear Overhauser effect (NOE) experiments. Application of the active compound to the seeds of S. gesnerioides at a concentration of 0.35 × 10⁻⁹ mol/disk led to 69% germination. The germination observed with application of GR-24, a positive control, at 0.57 × 10⁻¹⁰ mol/disk was 80%.     

MCM interference during licensing of DNA replication in Xenopus egg extracts-Possible Role of a C-terminal region of MCM3-

The minichromosome maintenance (MCM) complex, consisting of six subunits, Mcm2-7, is loaded onto replication origins through loading factors (origin recognition complex [ORC], Cdc6, and Cdt1) and forms an MCM double hexamer that licenses the initiation of DNA replication. Previous studies with Xenopus egg extracts showed that loading factors, especially Cdc6, dissociate from chromatin on MCM loading, but the molecular mechanism and physiological significance remain largely unknown. Using a cell-free system for MCM loading onto plasmid DNA in Xenopus egg extracts, we found that MCM loaded onto DNA prevents DNA binding of the loading factors ORC, Cdc6, and Cdt1. We further report that a peptide of the C-terminal region of MCM3 (MCM3-C), previously implicated in the initial association with ORC/Cdc6 in budding yeast, prevents ORC/Cdc6/Cdt1 binding to DNA in the absence of MCM loading. ATP-γ-S suppresses inhibitory activities of both the MCM loaded onto DNA and the MCM3-C peptide. Other soluble factors in the extract, but neither MCM nor Cdt1, are required for the activity. Conservation of the amino acid sequences of MCM3-C and its activity in vertebrates implies a novel negative autoregulatory mechanism that interferes with MCM loading in the vicinity of licensed origins to ensure proper origin licensing.     

Consequences of Sphaeropsis tip blight disease for the phytohormone profile and antioxidative metabolism of its pine host

Phytopathogenic fungi infections induce plant defence responses that mediate changes in metabolic and signalling processes with severe consequences for plant growth and development. Sphaeropsis tip blight, induced by the endophytic fungus Sphaeropsis sapinea that spreads from stem tissues to the needles, is the most widespread disease of conifer forests causing dramatic economic losses. However, metabolic consequences of this disease on bark and wood tissues of its host are largely unexplored. Here, we show that diseased host pines experience tissue dehydration in both bark and wood. Increased cytokinin and declined indole‐3‐acetic acid levels were observed in both tissues and increased jasmonic acid and abscisic acid levels exclusively in the wood. Increased lignin contents at the expense of holo‐cellulose with declined structural biomass of the wood reflect cell wall fortification by S. sapinea infection. These changes are consistent with H₂O₂ accumulation in the wood, required for lignin polymerization. Accumulation of H₂O₂ was associated with more oxidized redox states of glutathione and ascorbate pools. These findings indicate that S. sapinea affects both phytohormone signalling and the antioxidative defence system in stem tissues of its pine host during the infection process.     

Two Distinct Populations of Primary Cytotoxic Cells Infiltrating into Allografted Tumor Rejection Sites: Infiltration of Macrophages Cytotoxic against Allografted Tumor Precedes That of Multiple Sets of Cytotoxic T Lymphocytes with Distinct Specificity to Alloantigens

It has been reported that the rejection of tumor allografts is mainly mediated by cytotoxic T lymphocytes (CTLs). Here, we characterized the cytotoxic effector cells of C57BL/6 (B6; H-2^b) mice infiltrating into the rejection site of the i.p. allografted Meth A fibrosarcoma (or P815 mastocytoma) cells of H-2^d origin. Two types of cytotoxic cells (i.e., CD8⁺ CTLs and macrophages (Mφs)) were identified by flow cytometric fractionation of the infiltrates or by specific in vitro elimination of cells either with antibody (Ab)-coated beads or with an Ab-plus complement. Of particular interest, these effector cells showed distinct and unique target specificities. First, the CTLs were inactive against transplanted tumor (e.g., Meth A) cells, whereas they were cytotoxic against donor-related concanavalin A (Con A) blasts as well as CTLL-2 (H-2^b) cells transfected with a class I gene of H-2^d origin. A cold target competition assay suggested that the CTLs were composed of multiple sets of T cells, each of which specifically recognized different allo-antigens. Second, the Mφs lysed the allografted tumor cells but were inert toward the Con A blasts and the CTLL-2 transfectants. Unexpectedly, the infiltration of Mφs preceded the infiltration of CTLs by several days during the course of rejection. These results indicate that two distinct populations of unique cytotoxic cells (i.e., CTLs and Mφs) are induced in the allografted tumor rejection site, and that the infiltration of cytotoxic Mφs responsible for rejection precedes that of the CTLs cytotoxic against cells expressing donor-related allo-antigens.     

Accelerated dentinogenesis by inhibiting the mitochondrial fission factor,dynamin related protein 1

Undifferentiated odontogenic epithelium and dental papilla cells differentiate into ameloblasts and odontoblasts, respectively, both of which are essential for tooth development. These differentiation processes involve dramatic functional and morphological changes of the cells. For these changes to occur, activation of mitochondrial functions, including ATP production, is extremely important. In addition, these changes are closely related to mitochondrial fission and fusion, known as mitochondrial dynamics. However, few studies have focused on the role of mitochondrial dynamics in tooth development. The purpose of this study was to clarify this role. We used mouse tooth germ organ cultures and a mouse dental papilla cell line with the ability to differentiate into odontoblasts, in combination with knockdown of the mitochondrial fission factor, dynamin related protein (DRP)1. In organ cultures of the mouse first molar, tooth germ developed to the early bell stage. The amount of dentin formed under DRP1 inhibition was significantly larger than that of the control. In experiments using a mouse dental papilla cell line, differentiation into odontoblasts was enhanced by inhibiting DRP1. This was associated with increased mitochondrial elongation and ATP production compared to the control. These results suggest that DRP1 inhibition accelerates dentin formation through mitochondrial elongation and activation. This raises the possibility that DRP1 might be a therapeutic target for developmental disorders of teeth.     

Aminooxy‐naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l‐tryptophan aminotransferase: structure–activity relationships

We previously reported l ‐α‐aminooxy‐phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis, but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from AOPP to study the structure–activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These active compounds reduced the endogenous indole‐3‐acetic acid (IAA) content upon application to Arabidopsis seedlings. Among the compounds, we selected 2‐(aminooxy)‐3‐(naphthalen‐2‐yl)propanoic acid (KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169 showed typical auxin‐deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine ammonia‐lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly developed compounds constitute a class of inhibitors of TAA1. We designated them ‘pyruvamine’.