Cloning and characterization of novel ficolins from the solitary ascidian, Halocynthia roretzi

Ficolins are animal lectins with collagen-like and fibrinogen-like domains. They are involved in the first line of host defense against pathogens. Human ficolin/P35 as well as mannose-binding lectin (MBL) activates the complement lectin pathway in association with MBL-associated serine proteases. To elucidate the origin and evolution of ficolins, we separated approximately 40 kDa (p40) and approximately 50 kDa (p50) N-acetylglucosamine-binding lectins from hemolymph plasma of the solitary ascidian. Binding assays revealed that p40 recognizes N-acetyl groups in association with a pyranose ring and that p50 recognizes N-acetylglucosamine alone. Based on the amino acid sequences of the proteins, we isolated two clones each of p40 and p50 from the ascidian hepatopancreas cDNA and determined the entire coding sequences of these clones. Because all of the clones contained both collagen-like and fibrinogen-like domains, we concluded that these were homologs of the mammalian ficolin family and designated ascidian ficolins (AsFCNs). The fibrinogen-like domain of the AsFCNs shows 45.4-52.4% amino acid sequence identity with the mammalian ficolin family. A phylogenetic tree of the fibrinogen-like sequences shows that all the fibrinogen-like domains may have evolved from a common ancestor that branched off an authentic fibrinogen. These results suggest that AsFCNs play an important role with respect to ascidian hemolymph lectin activity and the correlation of different functions with binding specificity.     

The occurrence of two species of <Emphasis Type="Italic">Macroramphosus</Emphasis> (Gasterosteiformes: Macroramphosidae) in Japan: morphological and ecological observations on larvae,juveniles, and adults

Earlier opinions that Macroramphosus is monotypic are refuted, with two species apparently occurring in Japan (tentatively identified as M. gracilis and M. scolopax). In postsettlement young and adults, the former is characterized by a dark slender body (vs. red-orange and deep) and short second dorsal fin spine with a smooth posterior margin (vs. long spine with a serrated margin). Food habits also differ between the two species, which are either plankton or benthos feeders. Two types of Macroramphosus larvae and juveniles occurring at the surface were recognized, one having a straight ventral body profile of the body (identified here as M. gracilis) and the other having a notch in the anal region. The dark body of postsettlement M. gracilis is considered to be a retention of the character suited to the neustonic distribution of the larval and juvenile stages, the species remaining to ca. 40mm in standard length (SL) in that habitat (vs. to ca. 12mm SL in M. scolopax).     

Effect of estrogenic activity, and phytoestrogen and organochlorine pesticide contents in an experimental fish diet on reproduction and hepatic vitellogenin production in medaka (Oryzias latipes)

Endocrine-disrupting chemicals (EDCs) are giving rise to serious concerns for humans and wildlife. Phytoestrogens, such as daidzein and genistein in plants, and organochlorine pesticides are suspected EDCs, because their chemical structure is similar to that of natural or synthetic estrogens and they have estrogenic activity in vitro and in vivo. We assessed estrogenic activity and dietary phytoestrogen and organochlorine pesticide contents of various fish diets made in the United Kingdom, and compared them with those features of diets made in Japan that were tested in a previous study. Genistein and daidzein were detected in all of the diets. Using an in vitro bioassay, many of these diets had higher activation of estrogen beta-receptors than estrogen alpha-receptors. Organochlorine pesticides such as hexachlorobenzene, beta-benzene hexachloride (BHC), and gamma-BHC were detected in all fish diets. On the basis of these data, we investigated the effect of differing dietary phytoestrogen content in Japanese fish diets on hepatic vitellogenin production and reproduction (fecundity and fertility) in medaka (Oryzias latipes). Assessment of the effects of a 28-day feeding period on reproduction of paired medaka did not indicate significant differences in the number of eggs produced and fertility among all feeding groups. However, hepatic vitellogenin values were significantly higher for male medaka fed diet C (genistein, 58.5 +/- 0.6 microg/g; daidzein, 37.3 +/- 0.2 microg/g) for 28 days compared with those fed diet A (genistein, < 0.8 microg/g; daidzein, < 0.8 microg/g) or diet B (genistein, 1.4 +/- 0.1 microg/g; daidzein, 2.0 +/- 0.1 microg/g). Our findings indicate that fish diets containing high amounts of phytoestrogens, such as diet C, have the potential to induce hepatic vitellogenin production in male medaka, even if reproductive parameters are unaffected. Therefore, some diets, by affecting vitellogenin production in males, may alter estrogenic activity of in vivo tests designed to determine activity of test compounds added to the diet.     

Total synthesis of both enantiomers of dictyochromenol and their (Z)-isomers

Total syntheses of both enantiomers of dictyochromenol (1) and its (Z)-stereoisomer (2) were achieved with high enantiomeric purity. The results of this study reveal the relationship between the optical rotation of the resolved 1 enantiomers.     

Leaf-closing substance in Leucaena leucocephala

Potassium (2R,3R)-2,3,4-trihydroxy-2-methylbutanoate (1) was identified as a leaf-closing substance in the nyctinastic plant, Leucaena leucocephala. Compound 1 showed strong leaf-closing activity toward L. leucocephala and was not effective against other nyctinastic plants. The potassium ion was indispensable for the bioactivity of 1. Compound 1 gradually lost its bioactivity because of the exchange of the counter cation during isolation. A leaf-opening substance was also observed in the same plant.     

Developmental expression pattern of oncostatin M receptor beta in mice

Oncostatin M (OSM), which is predominantly expressed in bone marrow, is a member of the interleukin-6 family of cytokines, and appears to play important roles in hematopoiesis and the development of the liver. Recently, specific beta subunit of OSM receptor (OSMRbeta) was isolated from LO cells originated from aorta-gonad-mesonephros (AGM) region. In this study, we performed in situ hybridization to explore the expression pattern of OSMRbeta during murine embryogenesis, postnatal development, and in adult tissues. At 11.5 days postcoitum (dpc), the expression of OSMRbeta was first detected in aortic endothelial cells of the AGM region. At 14.5dpc, its gene expression was clearly observed in the primordia of some organs, including liver, thymus, choroid plexus, and limb, and persisted into postnatal mice. After birth, its gene expression became detectable in the other organs, such as lymph node, bone, heart, kidney, small intestine, nasal cavity, and lung.     

Phosphorylation of p27Kip1 on serine 10 is required for its binding to CRM1 and nuclear export

Phosphorylation of the cyclin-dependent kinase inhibitor p27(Kip1) has been thought to regulate its stability. Ser(10) is the major phosphorylation site of p27(Kip1), and phosphorylation of this residue affects protein stability. Phosphorylation of p27(Kip1) on Ser(10) has now been shown to be required for the binding of CRM1, a carrier protein for nuclear export. The p27(Kip1) protein was translocated from the nucleus to the cytoplasm at the G(0)-G(1) transition of the cell cycle, and this export was inhibited by leptomycin B, a specific inhibitor of CRM1-dependent nuclear export. The nuclear export and subsequent degradation of p27(Kip1) at the G(0)-G(1) transition were observed in cells lacking Skp2, the F-box protein component of an SCF ubiquitin ligase complex, indicating that these early events are independent of Skp2-mediated proteolysis. Substitution of Ser(10) with Ala (S10A) markedly reduced the extent of p27(Kip1) export, whereas substitution of Ser(10) with Asp (S10D) or Glu (S10E) promoted export. Co-immunoprecipitation analysis showed that CRM1 preferentially interacted with S10D and S10E but not with S10A, suggesting that the phosphorylation of p27(Kip1) on Ser(10) is required for its binding to CRM1 and for its subsequent nuclear export.     

Fourier transform infrared spectroscopy in high-pressure studies on proteins

Several aspects of the application of Fourier transform infrared spectroscopy (FTIR) in high-pressure studies on proteins are reviewed. Basic methodological considerations regarding spectral band assignments, quantitative analysis, and choice of pressure calibrants are also placed within the scope of this paper. This work attempts to evaluate recent developments in the field of high-pressure FTIR of proteins and its prospects for future. Particular attention is paid to the phenomenon of protein aggregation.