Expression of mouse Coiled-coil-DIX1 (Ccd1), a positive regulator of Wnt signaling, during embryonic development

Wnt signaling plays an important role in cell growth, differentiation, polarity formation, and neural development. We have recently identified the Coiled-coil-DIX1 (Ccd1) gene encoding a third type of a DIX domain-containing protein. Ccd1 forms homomeric and heteromeric complexes with Dishevelled and Axin, and positively regulates the Wnt/beta-catenin pathway. Here, we examined the spatiotemporal expression pattern of Ccd1 mRNA in mouse embryos from embryonic day 6.5 (E6.5) to E17.5 by in situ hybridization. Ccd1 expression was detected in the node region in gastrula embryos, in the cephalic mesenchyme and tail bud at E8.5, and in the branchial arch and forelimb bud at E9.5. In the central nervous system, Ccd1 expression began and persisted in the regions where the neurons differentiated, so that it was observed throughout the brain and spinal cord at E17.5. Ccd1 expression was also strong in the peripheral nervous system, including sensory cranial ganglia (trigeminal, facial, and vestibulocochlear ganglia), dorsal root ganglia, and autonomic ganglia (sympathetic ganglia, celiac ganglion, and hypogastric plexus). Ccd1 was detected in the sensory organs, such as the inner nuclear layer of the neural retina, saccule and cochlea of the inner ear, and nasal epithelium. Outside the nervous system, Ccd1 mRNA was observed in the cartilage, tongue, lung bud, stomach, and gonad at E12.5-E14.5, and in the tooth bud, bronchial epithelium, and kidney at E17.5. Taken together, these findings demonstrate that Ccd1 expression is observed in all the neurons in the nervous system, closely associated with neural crest-derived tissues, and largely overlapping with the regions where several Wnt genes are reported to play a role.     

Structure of 45,46,47-trinorhomoyessotoxin, a new yessotoxin analog, from Protoceratium reticulatum which represents the first detection of a homoyessotoxin analog in Japan

Contamination of yessotoxin (YTX) and its analogs in shellfish has occurred worldwide and has seriously damaged shellfish industries. One of the sources of YTX has been identified the dinoflagellate Protoceratium reticulatum. A new analog of YTX, 45,46,47-trinorhomoYTX, was isolated from cultures of the dinoflagellate P. reticulatum collected at Yamada Bay, Iwate in Japan. Its structure was determined by analysis of MS and NMR experiments. This is the first isolation and confirmation of a homoYTX analog in Japan.     

Upside-down spiders build upside-down orb webs: web asymmetry,spider orientation and running speed in Cyclosa

Almost all spiders building vertical orb webs face downwards when sitting on the hubs of their webs, and their webs exhibit an up–down size asymmetry, with the lower part of the capture area being larger than the upper. However, spiders of the genus Cyclosa, which all build vertical orb webs, exhibit inter- and intraspecific variation in orientation. In particular, Cyclosa ginnaga and C. argenteoalba always face upwards, and C. octotuberculata always face downwards, whereas some C. confusa face upwards and others face downwards or even sideways. These spiders provide a unique opportunity to examine why most spiders face downwards and have asymmetrical webs. We found that upward-facing spiders had upside-down webs with larger upper parts, downward-facing spiders had normal webs with larger lower parts and sideways-facing spiders had more symmetrical webs. Downward-facing C. confusa spiders were larger than upward- and sideways-facing individuals. We also found that during prey attacks, downward-facing spiders ran significantly faster downwards than upwards, which was not the case in upward-facing spiders. These results suggest that the spider''s orientation at the hub and web asymmetry enhance its foraging efficiency by minimizing the time to reach prey trapped in the web.     

Effective handling of induced-fit motion in flexible docking

For structure-based drug design, where various ligand structures need to be docked to a target protein structure, a docking method that can handle conformational flexibility of not only the ligand, but also the protein, is indispensable. We have developed a simple and effective approach for dealing with the local induced-fit motion of the target protein, and implemented it in our docking tool, ADAM. Our approach efficiently combines the following two strategies: a vdW-offset grid in which the protein cavity is enlarged uniformly, and structure optimization allowing the motion of ligand and protein atoms. To examine the effectiveness of our approach, we performed docking validation studies, including redocking in 18 test cases and foreign-docking, in which various ligands from foreign crystal structures of complexes are docked into a target protein structure, in 22 cases (on five target proteins). With the original ADAM, the correct docking modes (RMSD < 2.0 A) were not present among the top 20 models in one case of redocking and four cases of foreign-docking. When the handling of induced-fit motion was implemented, the correct solutions were acquired in all 40 test cases. In foreign-docking on thymidine kinase, the correct docking modes were obtained as the top-ranked solutions for all 10 test ligands by our combinatorial approach, and this appears to be the best result ever reported with any docking tool. The results of docking validation have thus confirmed the effectiveness of our approach, which can provide reliable docking models even in the case of foreign-docking, where conformational change of the target protein cannot be ignored. We expect that this approach will contribute substantially to actual drug design, including virtual screening.     

Intra-sexual Dimorphism in Male Mandibles and Male Aggressive Behavior in the Broad-Horned Flour Beetle Gnatocerus cornutus (Coleoptera: Tenebrionidae)

In the stored-product beetle, the broad-horned flour beetle, Gnatocerus cornutus (Fabricius), all males possess enlarged mandibles, widened gena, and a pair of small horns on the vertex, but females lack these completely. Observations of male-male interactions of G. cornutus showed that larger individuals won male-male fights, and that the mandibles were used as weapons. Morphological analysis based on the non-linearity test of Eberhard and Gutierrez's model (1991) showed that intra-sexual dimorphism in males was only found in the mandibles used in male-male combat, but not in the gena and horns. This beetle can be an ideal model for evolutionary studies of exaggerated weapons for male combat, because rearing successive generations and observing male fighting are easy.     

A protein associated with toll-like receptor 4 (PRAT4A) regulates cell surface expression of TLR4

TLRs recognize microbial products. Their subcellular distribution is optimized for microbial recognition. Little is known, however, about mechanisms regulating the subcellular distribution of TLRs. LPS is recognized by the receptor complex consisting of TLR4 and MD-2. Although MD-2, a coreceptor for TLR4, enhances cell surface expression of TLR4, an additional mechanism regulating TLR4 distribution has been suggested. We show here that PRAT4A, a novel protein associated with TLR4, regulates cell surface expression of TLR4. PRAT4A is associated with the immature form of TLR4 but not with MD-2 or TLR2. PRAT4A knockdown abolished LPS responsiveness in a cell line expressing TLR4/MD-2, probably due to the lack of cell surface TLR4. PRAT4A knockdown down-regulated cell surface TLR4/MD-2 on dendritic cells. These results demonstrate a novel mechanism regulating TLR4/MD-2 expression on the cell surface.     

Optimization of wastewater feeding for single-cell protein production in an anaerobic wastewater treatment process utilizing purple non-sulfur bacteria in mixed culture condition

Impacts of operation timing of feeding and withdrawal on anaerobic wastewater treatment utilizing purple non-sulfur bacteria have been investigated in mixed culture condition with acidogenic bacteria. Simulated wastewater containing glucose was treated in a laboratory-scale chemostat reactor, changing the timing of wastewater feeding and withdrawal. Rhodopseudomonas palustris, which does not utilize glucose as a substrate, was inoculated in the reactor. Rps. palustris was detected by a fluorescent in situ hybridization (FISH) technique using the specific Rpal686 probe. As a result, population ratios of Rps. palustris were over 20% through the operation. Rps. palustris could grow by utilizing metabolites of acidogenic bacteria that coexisted in the reactor. A morning feed was effective for a good growth of purple non-sulfur bacteria. A protein content of cultured bacteria was the highest when wastewater was fed in the morning. Dissolved organic carbon (DOC) removal was 94% independent of the timing control. Consequently, feeding in the morning is the optimum feed-timing control from the aspects of growth of purple non-sulfur bacteria and single-cell protein production.     

Evidence of cross-fertilization in a gonochoric population of the tadpole shrimp Triops numidicus (Crustacea: Branchiopoda: Notostraca)

Cross-fertilization was evident in a gonochoric population of the notostracan Triops numidicus, in which a thick, hard eggshell had been suspected of preventing the sperm from reaching the egg. Most of the eggs from copulated females hatched into larvae, but the eggs from virgin females did not develop. In the larvae, paternal DNA fragments were detected by AFLP. In histological sections, several spermatozoa were found in the shell of newly oviposited eggs in the brood pouches of copulated females, suggesting that the shell was still soft enough to be penetrated by spermatozoa. These results showed that copulation and subsequent fertilization achieved by penetration of sperm through the newly deposited eggshell were indispensable for the normal development of the eggs.     

The role of salicylic acid in the glutathione-mediated protection against photooxidative stress in rice

Salicylic acid (SA) is known to be an essential component responsible for disease resistance in dicotyledonous plants. In rice, however, tissue contains extremely high endogenous levels of SA that do not increase after pathogen infection, suggesting that the SA has other major functions in healthy leaves. Although involvement of SA in oxidative-stress response is known in some dicotyledonous plants, antioxidative role of SA in rice is obscure. In this study, we examined the involvement of SA in the protection against oxidative stress in rice, using transgenic plants expressing the bacterial nahG gene that encodes salicylate hydroxylase, an SA-degrading enzyme. In SA-deficient NahG rice, the glutathione pool size was constitutively diminished as compared with control plants. NahG seedlings showed a delayed development phenotype, an increased susceptibility to oxidative stress and they developed light-induced lesions in their leaves without pathogen infection. Conversely, treatment with an activator of the SA-mediated defense-signaling pathway, probenazole, increased the glutathione pool size and suppressed lesion formation. These results suggest that in rice, SA has an important role in the response to high-light-induced oxidative stress, through its regulatory effects on glutathione homeostasis.     

Effects of chloromethanes on growth of and deletion of the pce gene cluster in dehalorespiring Desulfitobacterium hafniense strain Y51

The dehalorespiring Desulfitobacterium hafniense strain Y51 efficiently dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by PceA reductive dehalogenase encoded by the pceA gene. In a previous study, we found that the significant growth inhibition of strain Y51 occurred in the presence of commercial cis-DCE. In this study, it turned out that the growth inhibition was caused by chloroform (CF) contamination of cis-DCE. Interestingly, CF did not affect the growth of PCE-nondechlorinating SD (small deletion) and LD (large deletion) variants, where the former fails to transcribe the pceABC genes caused by a deletion of the promoter and the latter lost the entire pceABCT gene cluster. Therefore, PCE-nondechlorinating variants, mostly LD variant, became predominant, and dechlorination activity was significantly reduced in the presence of CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride at 1 microM, but not carbon dichloride even at 1 mM.