piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus

Transgenic insects have been artificially produced to study functions of interesting developmental genes, using insect transposons such as piggyBac. In the case of the cricket, however, transgenic animals have not yet been successfully artificially produced. In the present study, we examined whether the piggyBac transposon functions as a tool for gene delivery in embryos of Gryllus bimaculatus. We used either a piggyBac helper plasmid or a helper RNA synthesized in vitro as a transposase source. An excision assay revealed that the helper RNA was more effective in early Gryllus eggs to transpose a marker gene of eGFP than the helper plasmid containing the piggyBac transposase gene driven by the G. bimaculatus actin3/4 promoter. Further, only when the helper RNA was used, somatic transformation of the embryo with the eGFP gene was observed. These results suggest that the piggyBac system with the helper RNA may be effective for making transgenic crickets.     

Macrophage Activation by Muramyl Dipeptide as Measured by Macrophage Spreading and Attachment

A synthetic bacterial cell wall constituent, muramyl dipeptide (MDP), was found to induce the enhancement of macrophage spreading and attachment on glass or plastic surfaces. Macrophages exposed to bacterial lipopolysaccharide or lymphokine-containing cell supernatants showed similar enhancement. This finding supports the view that MDP activates macrophages. MDP was also found to enhance the viability of macrophages but to inhibit ³H-thymidine incorporation by macrophages.     

Preparation and mass-spectral analysis of O-hydroxy-ethyl derivatives of -glucose

Various hydroxyethyl ethers of -glucose have been prepared in good yield by treating -glucose derivatives with 2-bromoethyl tetrahydropyranyl ether in the presence of sodium hydride. The derived O-(hydroxyethyl)- -glucitol acetates exhibited characteristic mass-spectral fragments. The furanose and pyranose forms of 1,2-O-ethylene- -glucose derived from 2-O-(2-hydroxyethyl)- -glucose were identified by mass-spectral analysis.     

Construction of a potato fraction library for the investigation of functional secondary metabolites

A potato fraction library was constructed to investigate functional secondary metabolites from 8 cultivars: Kitahime, Pilka, Sakurafubuki, Atlantic, Toyoshiro, Snowden, Kitamurasaki, and Northern Ruby, which were divided into flower, leaf, stem, roots, tuber peel, and tuber. Each fraction was a semi-purified extract and about 800 fractions were prepared for the library. They were analyzed by DAD-LC/MS to obtain structural information and were evaluated for various biological activities. LC/MS data showed that each part had a specific characteristic for their constituents supported by principal component analysis (PCA). Approximately 40% of fractions showed significant biological activities at 30 μg/mL, especially the flower fractions showed strong cytotoxicity. PCAs based on the activity and LC/MS data suggested that the strong cytotoxicity of flowers was derived from a complex mixture of potato glycoalkaloids. In addition, tuber peel fractions showed strong antimalarial activity, which had not been reported before. Also, some fractions showed significant antibacterial activities.     

Kinetic and structural studies on the catalytic role of the aspartic acid residue conserved in copper amine oxidase

Copper amine oxidase contains a post-translationally generated quinone cofactor, topa quinone (TPQ), which mediates electron transfer from the amine substrate to molecular oxygen. The overall catalytic reaction is divided into the former reductive and the latter oxidative half-reactions based on the redox state of TPQ. In the reductive half-reaction, substrate amine reacts with the C5 carbonyl group of the oxidized TPQ, forming the substrate Schiff base (TPQ(ssb)), which is then converted to the product Schiff base (TPQ(psb)). During this step, an invariant Asp residue with an elevated pKa is presumed to serve as a general base accepting the alpha proton of the substrate. When Asp298, the putative active-site base in the recombinant enzyme from Arthrobacter globiformis, was mutated into Ala, the catalytic efficiency dropped to a level of about 10(6) orders of magnitude smaller than the wild-type (WT) enzyme, consistent with the essentiality of Asp298. Global analysis of the slow UV/vis spectral changes observed during the reductive half-reaction of the D298A mutant with 2-phenylethylamine provided apparent rate constants for the formation and decay of TPQ(ssb) (k(obs) = 4.7 and 4.8 x 10(-4) s(-1), respectively), both of which are markedly smaller than those of the WT enzyme determined by rapid-scan stopped-flow analysis (k(obs) = 699 and 411 s(-1), respectively). Thus, Asp298 plays important roles not only in the alpha-proton abstraction from TPQ(ssb) but also in other steps in the reductive half-reaction. X-ray diffraction analyses of D298A crystals soaked with the substrate for 1 h and 1 week revealed the structures of TPQ(ssb) and TPQ(psb), respectively, as pre-assigned by single-crystal microspectrophotometry. Consistent with the stereospecificity of alpha-proton abstraction, the pro-S alpha-proton of TPQ(ssb) to be abstracted is positioned nearly perpendicularly to the plane formed by the Schiff-base imine double bond conjugating with the quinone ring of TPQ, so that the orbitals of sigma and pi electrons maximally overlap in the conjugate system. More intriguingly, the pro-S alpha proton of the substrate is released stereospecifically even in the reaction catalyzed by the base-lacking D298A mutant. On the basis of these results, we propose that the stereospecificity of alpha-proton abstraction is primarily determined by the conformation of TPQ(ssb), rather than the relative geometry of TPQ and the catalytic base.     

Examination of conditions inhibiting the formation of acrylamide in the model system of fried potato

Acrylamide (AAm) is produced in food through the reaction of asparagine and reducing sugar. We examined several methods of reducing the level of AAm using potato tubers. The fried model system that we employed consisted of thin slices that were first treated in water under different conditions before frying. A sufficient amount of water present in the fry material acts as an inhibitor against the formation of AAm and allows only a negligible amount of AAm to form. It was found that given the low content of water, the fry material temperature was sufficiently high to allow a relatively large level of AAm to form. Examination of water treatment prior to frying revealed that higher-temperature treatment water and longer treatment time resulted in the formation of lower levels of AAm. Moreover, removing some of the residual heat had an inhibiting effect on the formation of AAm.     

brachyenteron is necessary for morphogenesis of the posterior gut but not for anteroposterior axial elongation from the posterior growth zone in the intermediate-germband cricket Gryllus bimaculatus

In the long-germband insect Drosophila, all body segments and posterior terminal structures, including the posterior gut and anal pads, are specified at the blastoderm stage. In short- and intermediate-germband insects, however, posterior segments are sequentially produced from the posterior growth zone, a process resembling somitogenesis in vertebrates, and invagination of the posterior gut starts after anteroposterior (AP) axial elongation from the growth zone. The mechanisms underlying posterior segmentation and terminal patterning in these insects are poorly understood. In order to elucidate these mechanisms, we have investigated the roles of the Brachyury/brachyenteron (Bra/byn) homolog in the intermediate-germband cricket Gryllus bimaculatus. Loss-of-function analysis by RNA interference (RNAi) revealed that Gryllus byn (Gb'byn) is not required for AP axial elongation or normal segment formation, but is required for specification of the posterior gut. We also analyzed Gryllus caudal (Gb'cad) RNAi embryos using in situ hybridization with a Gb'byn probe, and found that Gb'cad is required for internalization of the posterior gut primordium, in addition to AP axial elongation. These results suggest that the functions of byn and cad in posterior terminal patterning are highly conserved in Gryllus and Drosophila despite their divergent posterior patterning. Moreover, because it is thought that the progressive growth of the AP axis from the growth zone, controlled by a genetic program involving Cdx/cad and Bra/byn, might be ancestral to bilaterians, our data suggest that the function of Bra/byn in this process might have been lost in insects.     

Synthesis of 11C-labeled 2-aminoethanol via a nitroaldol reaction using nitro[11C]methane

The nitroaldol reaction of nitro[¹¹C]methane and formaldehyde using EtOH and EtONa efficiently provided 2-nitro[¹¹C]ethanol in 3 min. The nitro group reduction in the presence of NiCl₂ and NaBH₄ in MeOH followed by purification using semi-preparative HPLC using 10% EtOH aqueous solution as an eluent proved to be a practical and accessible method for the synthesis of 2-amino[2-¹¹C]ethanol.