Identification of the functional expression of adenosine A<Subscript>3</Subscript> receptor in pancreas using transgenic mice expressing jellyfish apoaequorin

To investigate the functional expression of adenosine A₃ receptor (A3AR) in mammalian living tissues, we generated an apoaequorin-transgenic mouse that expresses jellyfish apoaequorin throughout its body. The expression of apoaequorin under the control of a strong CAG promoter was detected in various tissues, including the abdominal skin, adipose, ear, brain, esophagus, heart, inferior vena cava vessel, kidney, lens, liver, lung, pancreas, skeletal muscle, spleen, tail, testis, and thymus. The transgene was mapped to the C1–2 region of chromosome 16 by Fluorescence in situ hybridization analysis. Among these transgenic mouse tissues, we succeeded in detecting elevated responses of intracellular Ca²⁺ as a light emission of aequorin induced by the A3AR agonist in the pancreas, brain, and testis, the last two of which are known to be main tissues abundantly expressing A3AR. The A3AR agonist led to the phosphorylation of both extracellular signal-regulated kinase 1/2 and protein kinase B in mouse pancreas, and all the intracellular responses via A3AR were antagonized by the A3AR-specific antagonist. In addition, the mRNA expression of A3AR and the A3AR-induced intracellular responses were also found in the rat pancreatic acinar cell line AR42J. These results suggest that pancreas is one of the main tissues functionally expressing A3AR in mammalians in vivo, and that the present approach using transgenic mice that express apoaequorin throughout their bodies will facilitate the functional analysis of proteins of interest. Kazuya Yamano and Katsuhiro Mori contributed equally to this work     

Biological evaluation of de-O-sulfonated analogs of salacinol, the role of sulfate anion in the side chain on the alpha-glucosidase inhibitory activity

De-O-sulfonated analogs (10a, Y(-)=CH(3)OSO(3) and 10b, Y(-)=Cl) of salacinol, a naturally occurring glycosidase inhibitor, and its diastereomer (12a, Y(-)=CH(3)OSO(3)) with L-thiosugar moiety (1,4-dideoxy-1,4-epithio-L-arabinitol) were prepared. Their inhibitory activities against intestinal maltase and sucrase were examined and compared with those of the parent alpha-glycosidase inhibitor, salacinol (1a). Compounds 10a and 10b showed a potent inhibitory activity equal to that of 1a against both enzymes, although 12a was a weak inhibitor against sucrase and maltase. These results indicated that the O-sulfonate anion moiety of 1a is not essential for the inhibitory activity.     

The quail and chicken intestine have sialyl-galactose sugar chains responsible for the binding of influenza A viruses to human type receptors

The receptor specificity of influenza viruses is one factor that allows avian influenza viruses to cross the species barrier. The recent transmissions of avian H5N1 and H9N2 influenza viruses from chickens and/or quails to humans indicate that avian influenza viruses can directly infect humans without an intermediate host, such as pigs. In this study, we used two strains of influenza A virus (A/PR/8/34, which preferentially binds to an avian-type receptor, and A/Memphis/1/71, which preferentially binds to a human-type receptor) to probe the receptor specificities in host cells. Epithelial cells of both quail and chicken intestines (colons) could bind both avian- and human-type viruses. Infected cultured quail colon cells expressed viral protein and allowed replication of the virus strain A/PR/8/34 or A/Memphis/1/71. To understand the molecular basis of these phenomena, we further investigated the abundance of sialic acid (Sia) linked to galactose (Gal) by the alpha2-3 linkage (Siaalpha2-3Gal) and Siaalpha2-6Gal in host cells. In glycoprotein and glycolipid fractions from quail and chicken colon epithelial cells, there were some bound components of Sia-Gal linkage-specific lectins, Maackia amurensis agglutinin (specific for Siaalpha2-3 Gal) and Sambucus nigra agglutinin (specific for Siaalpha2-6Gal), indicating that both Siaalpha2-3Gal and Siaalpha2-6Gal exist in quail and chicken colon cells. Furthermore, we demonstrated by fluorescence high-performance liquid chromatography (HPLC) analysis that 5-N-acetylneuraminic acid was the main molecular species of Sia, and we demonstrated by multi-dimensional HPLC mapping and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis that bi-antennary complex-type glycans alpha2-6 sialylated at the terminal Gal residue(s) are major (more than 79%) sialyl N-glycans expressed by intestinal epithelial tissues in both the chicken and quail. Taken together, these results indicate that quails and chickens have molecular characterization as potential intermediate hosts for avian influenza virus transmission to humans and could generate new influenza viruses with pandemic potential.     

Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), was examined using COS-7 cells. 8-OH-dGTP and supF shuttle plasmid DNA were cointroduced by means of cationic liposomes, and the DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T-->C:G substitution mutations in the COS-7 cells. This result agrees with previous observations indicating that DNA polymerases misincorporate 8-OH-dGTP opposite A in vitro, and that the oxidized deoxyribonucleotide induces A:T-->C:G transversions in E. coli. These results constitute the first direct evidence to show that 8-OH-dGTP actually induces mutations in living mammalian cells.     

Production of 2-phenylethanol in roses as the dominant floral scent compound from L-phenylalanine by two key enzymes, a PLP-dependent decarboxylase and a phenylacetaldehyde reductase

We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[(2)H8] Phenylalanine was converted to [(2)H8] phenylacetaldehyde and [(2)H8]-2-phenylethanol by two enzymes derived from the flower petals of R. 'Hoh-Jun,' these being identified as pyridoxal-5'-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH3 together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.     

Influence of maceration temperature in red wine vinification on extraction of phenolics from berry skins and seeds of grape (Vitis vinifera)

The extraction of phenolics from berry skins and seeds of the grape, Vitis vinifera cv. Cabernet Sauvignon, during red wine maceration and the influence of different temperature conditions (cold soak and/or heating at the end of maceration) were examined. Phenolics contained mainly in berry skins, viz., anthocyanin, flavonol, and epigallocatechin units within proanthocyanidins, were extracted during the early stage of maceration, whereas those in seeds, viz., gallic acid, flavan-3-ol monomers, and epicatechin-gallate units within proanthocyanidins, were gradually extracted. In addition to their localization, the molecular size and composition of the proanthocyanidins possibly influenced the kinetics of their extraction. Cold soak reduced the extraction of phenolics from the seeds. Heating at the end of maceration decreased the concentration of proanthocyanidins. Thus, modification of the temperature condition during maceration affected the progress of the concentration of phenolics, resulting in an alteration of their make-up in the finished wine.     

Do immigrant female bonobos prefer older resident females as important partners when integrating into a new group?

Primates - Intergroup transfer is a critical part of the life history of group-living species, with considerable variation in its timings and patterns among species. Immigrant female bonobos are...     

Massilia sp. BS-1, a novel violacein-producing bacterium isolated from soil

A novel bacterium, Massilia sp. BS-1, producing violacein and deoxyviolacein was isolated from a soil sample collected from Akita Prefecture, Japan. The 16S ribosomal DNA of strain BS-1 displayed 93% homology with its nearest violacein-producing neighbor, Janthinobacterium lividum. Strain BS-1 grew well in a synthetic medium, but required both L-tryptophan and a small amount of L-histidine to produce violacein.     

Prediction of N-myristoylation modification of proteins by SVM

Attachment of a myristoyl group to NH(2)-terminus of a nascent protein among protein post-translational modification (PTM) is called myristoylation. The myristate moiety of proteins plays an important role for their biological functions, such as regulation of membrane binding (HIV-1 Gag) and enzyme activity (AMPK). Several predictors based on protein sequences alone are hitherto proposed. However, they produce a great number of false positive and false negative predictions; or they cannot be used for general purpose (i.e., taxon-specific); or threshold values of the decision rule of predictors need to be selected with cautiousness. Here, we present novel and taxon-free predictors based on protein primary structure. To identify myristoylated proteins accurately, we employ a widely used machinelearning algorithm, support vector machine (SVM). A series of SVM predictors are developed in the present study where various scales representing physicochemical and biological properties of amino acids (from the AAindex database) are used for numerical transformation of protein sequences. Of the predictors, the top ten achieve accuracies of >98% (the average value is 98.34%), and also the area under the ROC curve (AUC) values of >0.98. Compared with those of previous studies, the prediction accuracies are improved by about 3 to 4%.