Apis cerana japonica discriminates between floral color phases of the oriental orchid, Cymbidium floribundum

Foragers of the Japanese honeybee (Apis cerana japonica) were attracted by flowers of an oriental orchid (Cymbidium floribundum) and were observed to carry the pollinia on their scutella. After the removal of pollinia from the flowers, their labial color changed from white to reddish brown. Both artificial removal of pollinia and ethrel treatment of the flowers also induced this labial color change. Labia in color-changed flowers showed a decreased reflectance of wavelengths less than 670 nm compared to control intact flower. Both reflectance irradiance spectra and ultraviolet photographs showed that only the nectar guide in white (unchanged) flowers reflected ultraviolet light, and that this reflectance decreased with labial color change. Dual choice experiments showed that the honeybee foragers preferentially visited flowers having white labia rather than reddish brown. We suggest that Japanese honeybees discriminate between the floral phases of C. floribundum using color vision.     

Discovery of novel N-acylsulfonamide analogs as potent and selective EP3 receptor antagonists

A series of novel N-acylsulfonamide analogs were synthesized and evaluated for their binding affinity and antagonist activity for the EP3 receptor subtype. Representative compounds were also evaluated for their inhibitory effect on PGE₂-induced uterine contraction in pregnant rats. Among those tested, a series of N-acylbenzenesulfonamide analogs were found to be more potent than the corresponding carboxylic acid analogs in both the in vitro and in vivo evaluations. The structure activity relationships (SAR) are also discussed.     

Protein kinase C-δ is involved in induction of <Emphasis Type="Italic">NOX1</Emphasis> gene expression by aldosterone in rat vascular smooth muscle cells

In this study, we focused on the relationship between aldosterone and NOX1 expression in vascular smooth muscle cells (VSMCs). For the first time, with the use of specific inhibitors of protein kinase C (PKC), we report that PKCδ mediates upregulation of NOX1 induced by 10 nM aldosterone in cultured VSMCs. Participation of PKC in the mediation of NOX1 regulation was further confirmed by the effect of diacylglycerol, a PKC agonist, on the NOX1 RNA in A7r5 cells with Northern blot analysis. To establish cause and effect, we next silenced the PKCδ gene partly by RNA interference and found knockdown of PKCδ gene attenuated aldosterone-induced NOX1 expression, generation of superoxide, as well as protein synthesis in VSMCs. Taken together, these data indicated PKCδ might mediate aldosterone-dependent NOX1 upregulation in VSMCs. In addition, we showed that the cascade from aldosterone to PKCδ activation had the participation of the mineralocorticoid receptor.     

Combined genotoxic effects of radiation and a tobacco-specific nitrosamine in the lung of gpt delta transgenic mice

It is important to evaluate the health effects of low-dose-rate or low-dose radiation in combination with chemicals as humans are exposed to a variety of chemical agents. Here, we examined combined genotoxic effects of low-dose-rate radiation and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most carcinogenic tobacco-specific nitrosamine, in the lung of gpt delta transgenic mice. In this mouse model, base substitutions and deletions can be separately analyzed by gpt and Spi- selections, respectively. Female gpt delta mice were either treated with gamma-irradiation alone at a dose rate of 0.5, 1.0 or 1.5 mGy/h for 22 h/day for 31 days or combined with NNK treatments at a dose of 2 mg/mouse/day, i.p. for four consecutive days in the middle course of irradiation. In the gpt selection, the NNK treatments enhanced the mutation frequencies (MFs) significantly, but no obvious combined effects of gamma-irradiation were observable at any given radiation dose. In contrast, NNK treatments appeared to suppress the Spi- large deletions. In the Spi- selection, the MFs of deletions more than 1 kb in size increased in a dose-dependent manner. When NNK treatments were combined, the dose-response curve became bell-shaped where the MF at the highest radiation dose decreased substantially. These results suggest that NNK treatments may elicit an adaptive response that eliminates cells bearing radiation-induced double-strand breaks in DNA. Possible mechanisms underlying the combined genotoxicity of radiation and NNK are discussed, and the importance of evaluation of combined genotoxicity of more than one agent is emphasized.     

Evaluation of colors in green mutants isolated from purple bacteria as a host for colorimetric whole-cell biosensors

The change in carotenoid-based bacterial color from yellow to red can be applied to whole-cell biosensors. We generated several green mutants to emphasize the color change in such biosensors. The blue-green crtI-deleted mutant, Rhodopseudomonas palustris no.711, accumulated the colorless carotenoid precursor, phytoene. Green Rhodovulum sulfidophilum M31 accumulated neurosporene, a downstream product of phytoene. Another green mutant, Rhodobacter sphaeroides Ga, accumulated neurosporene and chloroxanthin, which are both downstream products of phytoene. All green mutants accumulated bacteriochlorophyll a. Photosynthetic membrane obtained from the green mutants all exhibited decreased absorption of wavelength range at 510–570 nm. Therefore, these indicate that the greenish bacterial colors were mainly caused by the existence of bacteriochlorophyll a and the changes in carotenoid composition in photosynthetic membrane. The colors of the green mutants and their wild-type strains were plotted in the CIE-L*a*b* color space, and the color difference (ΔE*ab) values between a green mutant and its wild type were calculated. ΔE*ab values were higher in the green mutants than in Rdv. sulfidophilum CDM2, the yellowish host strain of reported biosensors. These data indicate that change in bacterial color from green to red is more distinguishable than that from yellow to red as a reporter signal of carotenoid-based whole-cell biosensors.     

Crystal structures of blasticidin S deaminase (BSD): implications for dynamic properties of catalytic zinc

The set of blasticidin S (BS) and blasticidin S deaminase (BSD) is a widely used selectable marker for gene transfer experiments. BSD is a member of the cytidine deaminase (CDA) family; it is a zinc-dependent enzyme with three cysteines and one water molecule as zinc ligands. The crystal structures of BSD were determined in six states (i.e. native, substrate-bound, product-bound, cacodylate-bound, substrate-bound E56Q mutant, and R90K mutant). In the structures, the zinc position and coordination structures vary. The substrate-bound structure shows a large positional and geometrical shift of zinc with a double-headed electron density of the substrate that seems to be assigned to the amino and hydroxyl groups of the substrate and product, respectively. In this intermediate-like structure, the steric hindrance of the hydroxyl group pushes the zinc into the triangular plane consisting of three cysteines with a positional shift of approximately 0.6 A, and the fifth ligand water approaches the opposite direction of the substrate with a shift of 0.4 A. Accordingly, the zinc coordination is changed from tetrahedral to trigonal bipyramidal, and its coordination distance is extended between zinc and its intermediate. The shift of zinc and the recruited water is also observed in the structure of the inactivated E56Q mutant. This novel observation is different in two-cysteine cytidine deaminase Escherichia coli CDA and might be essential for the reaction mechanism in BSD, since it is useful for the easy release of the product by charge compensation and for the structural change of the substrate.     

Reduced Contamination by the Fusarium Mycotoxin Zearalenone in Maize Kernels through Genetic Modification with a Detoxification Gene

Maize is subject to ear rot caused by toxigenic Aspergillus and Fusarium species, resulting in contamination with aflatoxins, fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecene group and ZEN mycotoxins are produced by the cereal pathogen Fusarium graminearum. A transgenic detoxification system for the elimination of ZEN was previously developed using an egfp::zhd101 gene (gfzhd101), encoding an enhanced green fluorescent protein fused to a ZEN-degrading enzyme. In this study, we produced a transgenic maize line expressing an intact copy of gfzhd101 and examined the feasibility of transgene-mediated detoxification in the kernels. ZEN-degrading activity has been detected in transgenic kernels during seed maturation (for a period of 6 weeks after pollination). The level of detoxification activity was unaltered after an additional storage period of 16 weeks at 6°C. When the seeds were artificially contaminated by immersion in a ZEN solution for 48 h at 28°C, the total amount of the mycotoxin in the transgenic seeds was uniformly reduced to less than 1/10 of that in the wild type. The ZEN in the transgenic maize kernels was also efficiently decontaminated under conditions of lower water activity (a_w) and temperature; e.g., 16.9 μg of ZEN was removed per gram of seed within 48 h at an a_w of 0.90 at 20°C. F. graminearum infection assays demonstrated an absence of ZEN in the transgenic maize seeds, while the mycotoxin accumulated in wild-type kernels under the same conditions. Transgene-mediated detoxification may offer simple solutions to the problems of mycotoxin contamination in maize.     

Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-alpha antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL). TNF-alpha might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-kappaB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.     

Ligand-binding activity and expression profile of annexins in Caenorhabditis elegans

Mammalian annexins are implicated in several physiological mechanisms based on their calcium-dependent phospholipid/membrane binding and carbohydrate-binding activities. In this study, we investigated gene expression profiles of all four Caenorhabditis elegans annexins, nex-1, -2, -3 and -4, throughout the development, and compared phospholipid- and carbohydrate-binding properties of their protein products, NEX-1, -2, -3 and -4. We found that nex-1 and -3 are transcribed continuously during the developmental stages, while expression of nex-2 and -4 appeared to be temporal, peaking at the L1 stage followed by a gradual decrease toward the adult stage. NEX-1 and -3 were detected as single protein band in total worm extracts by immunoblotting, but NEX-2 was heterogenic in size. NEX-1, -2, and -3 showed the binding activities to phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine, but not to phosphatidylcholine. In contrast to their uniform phospholipids-binding properties, their glycosaminoglycan-binding activities were distinctive. NEX-2 bound to heparan sulfate and chondroitin, NEX-3 bound only to heparan sulfate, and NEX-1 showed no lectin activities under tested conditions. NEX-4 had neither phospholipids- nor carbohydrate-binding properties. Differentiated expression profiles and ligand-binding properties of NEX-1, -2, -3 and -4, shown in our study, may represent distinctive roles for each C. elegans annexins.