Competition for Space Is Controlled by Apoptosis-Induced Change of Local Epithelial Topology

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Identification,Characterization, and Mapping of a Novel SNP Associated with Body Color Transparency in Juvenile Red Sea Bream (<Emphasis Type="Italic">Pagrus major</Emphasis>)

We previously reported a body color deformity in juvenile red sea bream, which shows transparency in the juvenile stage because of delayed chromatophore development compared with normal individuals, and this finding suggested a genetic cause based on parentage assessments. To conduct marker-assisted selection to eliminate broodstock inheriting the causative gene, developing DNA markers associated with the phenotype was needed. We first conducted SNP mining based on AFLP analysis using bulked-DNA from normal and transparent individuals. One SNP was identified from a transparent-specific AFLP fragment, which significantly associated with transparent individuals. Two alleles (A/G) were observed in this locus, and the genotype G/G was dominantly observed in the transparent groups (97.1%) collected from several production lots produced from different broodstock populations. A few normal individuals inherited the G/G genotype (5.0%), but the A/A and A/G genotypes were dominantly observed in the normal groups. The homologs region of the SNP was searched using a medaka genome database, and intron 12 of the Nell2a gene (located on chromosome 6 of the medaka genome) was highly matched. We also mapped the red sea bream Nell2a gene on the previously developed linkage maps, and this gene was mapped on a male linkage group, LG4-M. The newly found SNP was useful in eliminating broodstock possessing the causative gene of the body color transparency observed in juvenile stage of red sea bream.     

Characterization of thermostable FMN-dependent NADH azoreductase from the moderate thermophile Geobacillus stearothermophilus

The gene encoding an FMN-dependent NADH azoreductase, AzrG, from thermophilic Geobacillus stearothermophilus was cloned and functionally expressed in recombinant Escherichia coli. Purified recombinant AzrG is a homodimer of 23 kDa and bore FMN as a flavin cofactor. The optimal temperature of AzrG was 85 °C for the degradation of Methyl Red (MR). AzrG remained active for 1 h at 65 °C and for 1 month at 30 °C, demonstrating both superior thermostability and long-term stability of the enzyme. AzrG efficiently decolorized MR, Ethyl Red at 30 °C. Furthermore, the enzyme exhibited a wide-range of degrading activity towards several tenacious azo dyes, such as Acid Red 88, Orange I, and Congo Red. These results suggested the sustainable utilization of G. stearothermophilus as an azo-degrading strain for AzrG carrying whole-cell wastewater treatments for azo pollutants under high temperature conditions.     

Synthesis and degradation of long-chain base phosphates affect fumonisin B<Subscript>1</Subscript>-induced cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Fumonisin B₁ (FB₁), an inducer of cell death, disrupts sphingolipid metabolism; large accumulations of de novo synthesized free long-chain bases (LCBs) are observed. However, it remains unclear whether tolerance to FB₁ toxicity in plants is connected with preventing the accumulation of free LCBs through their phosphorylation. Here a workflow for the extraction, detection and quantification of LCB phosphates (LCBPs) in Arabidopsis thaliana was developed. We studied the effect of expression of genes for three enzymes involved in the synthesis and degradation of LCBPs, LCB kinase (LCBK1), LCBP phosphatase (SPP1) and lyase (DPL1) on FB₁-induced cell death. As expected, large accumulations of saturated free LCBs, dihydrosphingosine and phytosphingosine, were observed in the FB₁-treated leaves. On the other hand, a high level of sphingenine phosphate was found in the FB₁-treated leaves even though free sphingenine was found in low amounts in these leaves. In comparison of WT and spp1 plants, the LCBP/LCB ratio is likely to be correlated with the degree of FB₁-induced cell death determined by trypan blue staining. The FB₁-treated leaves in dpl1 plants showed severe cell death and the elevation of free LCBs and LCBPs. LCBK1-OX and -KD plants showed resistance and sensitivity to FB₁, respectively, whereas free LCB and LCBP levels in FB₁-treated LCBK1-OX and -KD plants were moderately different to those in FB₁-treated WT plants. Overall, the findings described here suggest that LCBP/LCB homeostasis is an important topic that participates in the tolerance of plant cells to FB₁.     

(-)-hydroxycitrate ingestion increases fat oxidation during moderate intensity exercise in untrained men

We examined the effects of (-)-Hydroxycitrate (HCA) ingestion on fat oxidation during moderate intensity exercise in untrained men. Six subjects ingested 500 mg of HCA or a placebo for 5 days and did endurance exercise. Blood FFA concentrations were significantly increased and respiratory exchange ratio (RER) decreased by HCA ingestion. These results suggested short-term HCA ingestion increases fat oxidation in untrained men.     

Genetic structure of the clonal herb Tanakaea radicans (Saxifragaceae) at multiple spatial scales,revealed by nuclear and mitochondrial microsatellite markers

The genus Tanakaea is a plant genus that consists of one or two evergreen herbaceous species in Japan and China. As rithophytic plant species occur on shaded rocks, the populations are usually isolated and sporadically found in disjunct areas. To evaluate the genetic structure of the species at multiple spatial scales, 10 nuclear and mitochondrial microsatellite markers were developed. The novel markers showed high genetic variations (two to 15 alleles and H_e from 0.400 to 0.894). Clonal samples were identified with the probability of identity of 9.0E‐8. When evaluated with 11 populations in Japan, significant genetic differentiation between regional population groups was detected (F_ST = 0.313 between Shikoku and Honshu islands), suggesting they have long been isolated from each other. Overall, these markers will be useful for population genetic research to investigate clonal structure and genetic diversity and levels of genetic differentiation between the geographically isolated populations.     

Antimalarial effect of bis-pyridinium salts, N,N'-hexamethylenebis(4-carbamoyl-1-alkylpyridinium bromide)

The in vitro antimalarial activity of bis-pyridinium salts, N,N'-hexamethylenebis(4-carbamoyl-1-decylpyridinium bromide) and their derivatives, against the Plasmodium falciparum FCR-3 strain (ATCC 30932, chloroquine-sensitive) was evaluated. All test compounds exhibited antimalarial activity over a concentration range of 3.5microM to 10nM. The chain length of the N1-alkyl moiety was found to be very beneficial in terms of antimalarial activity, and in this series of compounds, the most appropriate N1-alkyl chain length was found to be eight.     

Enzymatic properties of Myxococcus xanthus exopolyphosphatases mxPpx1 and mxPpx2

Myxococcus xanthus possesses two exopolyphosphatases, mxPpx1 and mxPpx2, which belong to the family of Ppx/GppA phosphatases; however, their catalytic properties have not been described. mxPpx1 and mxPpx2 contain 311 and 505 amino acid residues, respectively; mxPpx2 has an additional C-terminal region, which corresponds to the metal-dependent HDc phosphohydrolase domain. mxPpx1 mainly hydrolyzed short-chain polyPs (polyP₃ and polyP₄), whereas mxPpx2 preferred long-chain polyP_60–70 and polyP_700–1000. mxPpx2 was activated by 25–50 mM KCl, but mxPpx1 did not significantly depend on K⁺. In addition, mxPpx1 and mxPpx2 showed weak hydrolysis of ATP and GTP in the absence of K⁺, and mxPpx2 could also hydrolyze guanosine pentaphosphate (pppGpp) in the presence of K⁺. The exopolyphosphatase activity of mxPpx1 toward polyP₃ was inhibited by polyP_700–1000 and that of mxPpx2 toward polyP_60–70 and polyP_700–1000, by pyrophosphate. To clarify the function of the mxPpx2 C-terminal domain, it was fused to mxPpx1 (mxPpx1-2C) and deleted from mxPpx2 (mxPpx2?C). Compared to wild-type mxPpx2, mxPpx2?C had significantly reduced exopolyphosphatase activity toward long-chain polyPs (by 90%), whereas that toward polyP₃ and polyP₄ was much less affected; furthermore, the phosphohydrolase activity toward pppGpp, ATP, and GTP was also decreased (by 30–75%). In contrast, mxPpx1-2C had increased hydrolytic activity compared to mxPpx1. Furthermore, mxPpx2?C lost the requirement for K⁺ characteristic for the wild-type enzyme, whereas mxPpx1-2C acquired it. These results suggest that the C-terminal domain of mxPpx2 is necessary for its maximum hydrolytic activity, especially toward long-chain polyPs, and defines mxPpx2 dependency on K⁺ for activation.     

Visualization of abscisic acid-perception sites on the plasma membrane of stomatal guard cells

Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).