Studies of novel bioactive substances in the spent media of cell lines using protein-free culture system]

Many human cell lines have been maintained in fetal bovine serum (FBS)-supplemented medium. These produce and secrete many substances such as transferrin, alpha 1-antitrypsin, alpha 2-macroglobulin, alkaline phosphatase, gamma-glutamyltranspeptidase, creatine kinase, carcino-embryonic antigen, alpha-fetoprotein, and cytokines including colony stimulating factors and transforming growth factors and further they may produce small amounts of unknown substances. Usually, small amounts of substances have to be concentrated as highly as possible for detection, but FBS interferes with procedures. A protein-free culture system in an ideal method for detecting small quantities of substances which originate from cell lines without interference by FBS. Our protein-free culture system can be available in every laboratory since this is not only an economical method, but also an effective method for the saving of purification procedures. Moreover, this is a most suitable method for surveying unknown substances derived from cell lines.     

Immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) as a marker of interferon therapy in patients with persistent hepatitis B virus infection

Immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) was measured by radioimmunoassay in the sera of 96 HBV carriers. IgM anti-HBc was detected in 17 of 66 patients with chronic active hepatitis and in 4 of 11 with liver cirrhosis. This antibody was not present in asymptomatic carriers or in patients with chronic persistent hepatitis. Testing of sequential samples revealed that the presence of IgM anti-HBc indicated active replication of HBV and at the same time an immune response to the virus. The relationship between IgM anti-HBc and the response to interferon (IFN) therapy was also studied. Results showed that IgM anti-HBc is a useful marker of the efficacy of interferon therapy.     

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.     

Changes in abscisic acid levels during dormancy release in freesia corms

A high level of free-abscisic acid (ABA) was detected when corms were still in deep dormancy. The level of free-ABA decreased as the corm dormancy disappeared and increased temporarily after complete release from dormancy. A gradual slight increase of bound-ABA was observed during dormancy release.Treatment of dormant corms with benzyladenine (BA) increased sprouting but the sprouts did not show normal growth. Ethylene treatment induced complete sprouting and subsequent normal growth. Changes in ABA levels and ethylene production are discussed in relation to dormancy release in freesia corms.     

2',4'-BNA(NC): a novel bridged nucleic acid analogue with excellent hybridizing and nuclease resistance profiles

Oligonucleotides modified with 2 ',4 '-BNA(NC) (N-H)/(N-Me) monomers exhibited excellent hybridizing and nuclease resistance properties. Duplex and triplex thermal stabilities were greatly enhanced by incorporating 2',4'-BNA(NC) (N-H) and (N-Me) monomers and nuclease resistance was tremendously higher than that of natural oligonucleotide.     

IL-17 is involved in bone resorption in mouse periapical lesions

Periapical lesions are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. Although various immunological studies concerning periapical bone resorption have been reported, the role of cytokines in the formation of periapical lesions remains unclear. In this study, the role of IL-17A in periapical lesions in mice was investigated. Normal C57BL/6, IFN-γ^−/−, TNF-α^−/−, and IL-17A^−/− mice were subjected to pulp exposure and infected with Prevotella intermedia (ATCC25611) and Porphyromonas gingivalis (ATCC33277) in the mandibular first molar. Periapical lesions were determined by μCT on day 21 after infection, and 3D visual construction was performed using 3D picture quantification software. The expression of IL-17A mRNA in periapical lesions was determined by the RT-PCR and real-time RT-PCR method. Periapical lesions developed in wild-type, IFN-γ^−/−, and TNF-α^−/− mice after infection with P. intermedia and P. gingivalis . However, periapical lesions were not observed in IL-17A^−/− mice. The expression of IL-17A mRNA was significantly induced in periapical lesions of wild-type mice after infection. These results suggest that IL-17A, but not IFN-γ or TNF-α, plays an important role in the formation of periapical lesions.