Computational analysis and functional expression of ancestral copepod luciferase

We recently reported the cDNA sequences of 11 copepod luciferases from the superfamily Augaptiloidea in the order Calanoida. They were classified into two groups, Metridinidae and Heterorhabdidae/Lucicutiidae families, by phylogenetic analyses. To elucidate the evolutionary processes, we have now further isolated 12 copepod luciferases from Augaptiloidea species (Metridia asymmetrica, Metridia curticauda, Pleuromamma scutullata, Pleuromamma xiphias, Lucicutia ovaliformis and Heterorhabdus tanneri). Codon-based synonymous/nonsynonymous tests of positive selection for 25 identified copepod luciferases suggested that positive Darwinian selection operated in the evolution of Heterorhabdidae luciferases, whereas two types of Metridinidae luciferases had diversified via neutral mechanism. By in silico analysis of the decoded amino acid sequences of 25 copepod luciferases, we inferred two protein sequences as ancestral copepod luciferases. They were expressed in HEK293 cells where they exhibited notable luciferase activity both in intracellular lysates and cultured media, indicating that the luciferase activity was established before evolutionary diversification of these copepod species.     

Establishment and characterization of monoclonal antibodies against Chuzan virus K-47

We established sixteen mouse monoclonal antibodies reactive to Chuzan virus K-47 strain using P3-X63-Ag8-U1 cells as fusion partner cells. Among them, CG53/2/4 recognized a 100K structural protein of the virus. The 100K antigen lost it's antigenicity for CG53/2/4 after mild periodate oxidation treatment, suggesting that the 100K viral antigen is a glycoprotein. In addition, CG53/2/4 neutralized the viral infectivity. This indicates that the 100K glycoprotein is essential for the infection of the virus. The other monoclonal antibodies reacted with a 41K antigen of the virus. Especially CG1/1 showed the highest reactivity to the virus. Forward step sandwich assay using CG1/1 and biotinylated CG53/2/4 could detect the virus at 10TCID₅₀/ml. Therefore, these monoclonal antibodies can evantually predict the virus infection to the animals before their sideration.     

Balanced ubiquitylation and deubiquitylation of Frizzled regulate cellular responsiveness to Wg/Wnt

Wingless (Wg)/Wnt has been proposed to exert various functions as a morphogen depending on the levels of its signalling. Therefore, not just the concentration of Wg/Wnt, but also the responsiveness of Wg/Wnt‐target cells to the ligand, must have a crucial function in controlling cellular outputs. Here, we show that a balance of ubiquitylation and deubiquitylation of the Wg/Wnt receptor Frizzled determines the cellular responsiveness to Wg/Wnt both in mammalian cells and in Drosophila, and that the cell surface level of Frizzled is regulated by deubiquitylating enzyme UBPY/ubiquitin‐specific protease 8 (USP8). Although ubiquitylated Frizzled underwent lysosomal trafficking and degradation, UBPY/USP8‐dependent deubiquitylation led to recycling of Frizzled to the plasma membrane, thereby elevating its surface level. Importantly, a gain and loss of UBPY/USP8 function led to up‐ and down‐regulation, respectively, of canonical Wg/Wnt signalling. These results unveil a novel mechanism that regulates the cellular responsiveness to Wg/Wnt by controlling the cell surface level of Frizzled.     

Restoration of Mismatch Repair Functions in Human Cell Line Nalm-6, Which Has High Efficiency for Gene Targeting

Gene targeting is a powerful approach in reverse genetics. The approach has been hampered in most of human cell lines, however, by the poor targeting efficiency. Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, exhibits exceptionally high gene targeting efficiency and is used in DNA repair and the related research fields. Nonetheless, usage of the cell line is still limited partly because it lacks expression of MSH2, a component of mismatch repair complex, which leads to increased genome instability. Here, we report successful restoration of MSH2 expression in Nalm-6 cells and demonstrate that the recovery does not affect the high targeting efficiency. We recovered the expression by introduction of cDNA sequences corresponding to exons 9 to 16 at downstream of exon 8 of the MSH2 gene. Endogenous exons 9 to 16 were deleted in the cell line. The MSH2 expression substantially reduced spontaneous HPRT mutation frequency. Moreover, gene targeting efficiency in the MSH2-expressing cells was similar to that in the MSH2-lacking cells. In fact, we generated heterozygously REV3L knockout and the catalytically dead mutants in the MSH2-proficient Nalm-6 cells with efficiency of 20–30%. The established cell line, Nalm-6-MSH+, is useful for reverse genetics in human cells.     

The role of the brown-eared bulbulHypsypetes amaurotis as a seed dispersal agent

The role of the brown-eared bulbul,Hypsypetes amaurotis, as a dispersal agent for seeds of fruiting plants was studied by field observations for two years, in parallel with laboratory experiments on seed germination. Bulbuls consumed fruits of 53 species from 24 plant families. The fruits of these plants had similar color and size, and these characteristics were likely to enhance the feeding efficiency of the frugivore. Laboratory experiments on 20 food plant species demonstrated that: (1) no seeds were injured by passing through the bulbul's gut; (2) seeds that had passed through the bulbul's gut were still able to germinate and (3) fruit pulp reduced germination ability. When pulp was removed by passing through bulbul's gut, or by hand, germination was improved. An estimate of the home range of six bulbuls suggested that they may transport seeds for at least 300 m.     

Suanzaorentang, and anxiolytic Chinese medicine, affects the central adrenergic and serotonergic systems in rats

Previously, we found that the ancient Chinese remedy of Suanzaorentang was a promising anxiolytic drug (Chen and Hsieh, 1985; Chen and Hsieh, 1985a). We also found that Suanzaorentang decreased the turnover rate of central monoamines and central catecholaminergic activity (Hsieh, et al., 1986). In this study, we found that 5-hydroxytryptophan (5-HTP) induced decrease in locomotor activity was significantly antagonized by Suanzaorentang, p-chlorophenylalanine (p-CPA) induced increase in locomotor activity was significantly inhibited by Suanzaorentang, Suanzaorentang had no significant effects on baclofen, muscimol, aminooxyacetic acid (AOAA) and thiosemicarbazide induced changes in locomotor activity, Suanzaorentang significantly decreased vanillylmandelic acid (VMA) in striatum and hippocampus, homovanillic acid (HVA) in hippocampus and 5-hydroxyindol acetic acid (5-HIAA) in striatum and hypothalamus, Suanzaorentang significantly reversed the alpha-methyltyrosine (alpha-MT) produced decrease in DA concentrations in striatum and hippocampus, and (6) Suanzaorentang significantly reversed the p-CPA produced decrease in 5-HT concentrations on amygdala. These facts implied that Suanzaorentang might decrease the serotonergic activity but have no significant effect on GABAergic activity. The main locus of action might be in the limbic system.     

Changes in β-Galactosidase activity during differentiation and dedifferentiation inDictyostelium discoideum

β-Galactosidase (EC 3.2.1.23) ofDictyostelium discoideum was investigated for its properties and activity during differentiation and dedifferentiation. β-Galactosidase of this organism had a pH optimum at 3.5. The specific activity of this enzyme was increased gradually from the time of initiation of differentiation and reached a peak at the aggregation stage. Then the activity of the enzyme showed a slight decrease followed by a further increase and reached a maximum at early culmination. During dedifferentiation of cells disaggregated from a slug, the activity of the enzyme was increased, reached a maximum after 3 hr of incubation and then decreased nearly to the original level of activity after completion of dedifferentiation. This increase in the enzyme activity coincided with decomposition of acid mucopolysaccharide contained in the prespore specific vacuoles, and both processes were sensitive to cycloheximide. No increase in the activity of acetylglucosaminidase (EC 3.2.1.30), another lysosomal enzyme, was observed during the process. Possible roles of β-galactosidase in cell type conversion as well as in dedifferentiation of the prespore cell were discussed.