Tsutomu Takegami

A novel clinical entity, IgG4-related disease (IgG4RD): general concept and details

IgG4-related disease (IgG4RD) is a novel clinical disease entity characterized by elevated serum IgG4 concentration and tumefaction or tissue infiltration by IgG4-positive plasma cells. IgG4RD may be present in a certain proportion of patients with a wide variety of diseases, including Mikulicz’s disease, autoimmune pancreatitis, hypophysitis, Riedel thyroiditis, interstitial pneumonitis, interstitial nephritis, prostatitis, lymphadenopathy, retroperitoneal fibrosis, inflammatory aortic aneurysm, and inflammatory pseudotumor. Although IgG4RD forms a distinct, clinically independent disease category and is attracting strong attention as a new clinical entity, many questions and problems still remain to be elucidated, including its pathogenesis, the establishment of diagnostic criteria, and the role of IgG4. Here we describe the concept of IgG4RD and up-to-date information on this emerging disease entity.

Molecular cloning and complete nucleotide sequence of the genome of Japanese encephalitis virus Beijing-1 strain

The genomic RNA of the Japanese encephalitis virus (JEV) Beijing-1 strain was reversely transcribed and the synthesized cDNA was molecularly cloned. Six continuous cDNA clones that cover the entire virus genome were established and sequenced to determine the complete nucleotide sequence of the JEV RNA. The precise genomic size was estimated as 10,965 bases long. With flanking 95 bases at the 5′ and 583 bases at the 3′ non-coding regions, one long open reading frame (ORF) was revealed encoding a virus polyprotein with 3,429 amino acid residues. Because of sequence homologies observed between JEV and other flaviviruses, the genome organization of JEV appears to be identical with other flaviviruses. Genetic variation detected among flavivirus genomes is consistent with the established serological relatedness between JEV and other members of flaviviruses. The secondary structure of the JEV genome is deduced and discussed concerning its involvement in genome replication.

Ionic liquid enables simple and rapid sample preparation of human culturing cells for scanning electron microscope analysis

Ionic liquid is a kind of salt that stays in a molten state even at room temperature. It does not vaporize at all in vacuum and facilitates electrical conductivity to the sample surfaces for observations with a scanning electron microscope (SEM). In this study, we used an ionic liquid in SEM for the first time to observe fixed human culture cells. The condition for the cell culture using wrapping sheets and SEM settings were varied to elucidate the optimized protocol. Compared to samples prepared by the conventional way, the ionic liquid‐treatment of samples gave SEM images of the cellular ultra structures in more detail, enabling observation of microvilli that made bridges between separated cells. In addition, the ionic liquid treatment is less time consuming as well as less laborious compared with the conventional way that includes dehydration, drying, and conductivity treatments. Totally, we concluded the ionic liquid is a useful reagent for SEM sample preparation. Microsc. Res. Tech., 2011.

Inhibitory effect of RNAi on Japanese encephalitis virus replication in vitro and in vivo.

Flaviviruses include many insect‐mediated small viruses and still cause serious problems in the world. In humans, JEV can cause acute meningioencephalomyelitis, resulting in fatality rates of 5 to 40%. RNA‐interference (RNAi) as an antiviral mechanism was originally discovered in plants and then found in the specific suppression of gene expression of other organisms such as Caenorhabditis elegans, Drosophila and vertebrates. As JEV is an RNA virus, RNAi could be a reasonable approach for therapeutic purposes to use against Japanese encephalitis. In this study, we examined the effect of RNAi on JEV replication. Viral reproduction in Vero cells was decreased to 7.2% and 39.0% of control by the transfection of small interference RNAs, JCR and JN3R at 250 NM, respectively. Under the transfection of 5 μg/ml pJRi which produces stem‐loop RNAi, viral reproduction was decreased to about 10% of control. Western blot analysis indicated that RNAi inhibited the translation level. We used pJRi in the animal experiment. After the inoculation of viruses at 5× 103 PFU, pJRi at 1.0 and 5.0 μg/g was injected into mice i.p. JEV‐infected control mice (n=5) died within 15 days. pJRi (1.0 or 5.0 μg/g)‐medicated mice survived 40 or 80% at 15 days. The data clearly indicate that pJRi has highly potent inhibitory activity against JEV replication in vivo. The results in vivo and in vitro provide evidence that JEV replication was efficiently inhibited by RNAi and RNAis could be used as an antiviral drug against JEV infection.

Molecular characterization of the Japanese encephalitis virus representative immunotype strain JaGAr 01

We determined the full genomic sequence of the Japanese encephalitis virus JaGAr 01 strain and its predicted amino acid sequence. Nucleotide sequence comparison with ten fully sequenced JE strains shows a homology range from 89.62 to 99.49%. Amino acid sequence homologies range from 96.85 to 99.74%. Comparison of amino acid sequences shows a unique amino acid, arginine, for JaGAr 01 at position 123 of the E-protein, while the eight other strains contained serine. Secondary structure prediction by free energy minimization shows a unique structure for JaGAr 01 that includes an RNA segment that is conserved for all flaviviruses. Speculation is made about the role these results may play in the replication and antigenic characteristics of JaGAr 01. Phylogenetic analyses of the E-protein of JaGAr 01 together with 35 other JE strains showed diversity in amino acid characteristics between the prototype strains Nakayama, JaGAr 01 and Beijing-1. Phylogenetic trees computed by neighbor joining and Fitch Margoliash analysis of nucleic acid and protein sequences showed Nakayama and Beijing in one cluster different from JaGAr 01.

Localization and functions of Japanese encephalitis virus nonstructural proteins NS3 and NS5 for viral RNA synthesis in the infected cells.

Recently it has been reported that Japanese encephalitis virus (JEV)‐specific RNAs can be synthesized in vitro in the subcellular fraction including outer‐nuclear membrane (Takegami and Hotta, 1989). The results of Western blot analysis and indirect immunofluorescence test using two kinds of monospecific antisera against JEV nonstructural proteins NS3 and NS5 showed that NS3 and NS5 were membrane‐associated proteins and formed the complex at the perinuclear site in the infected cells. Both antisera against NS3 and NS5 inhibited in vitro RNA synthesis. These results suggest that NS5 and NS3 play important role(s) in flavivirus RNA replication.

Japanese encephalitis virus nonstructural protein NS3 has RNA binding and ATPase activities

Sequence data suggest that Japanese encephalitis virus (JEV) protein NS3 is a multifunctional protein with sequence motifs characteristic of a protease and a helicase. To examine the functions of JEV-NS3, a fusion protein of NS3 inEscherichia coli was generated. Analysis by Western blot using monospecific rabbit antisera generated against the fusion protein (anti-MBJEN3) showed that NS3 was localized in the membrane fraction of JEV-infected cells and the particulate fraction of bacteria extracts. The addition of anti-MBJEN3 sera reduced JEV-specific RNA synthesis activity in a in vitro system. In addition, NS3 was shown to exhibit RNA binding and ATPase activities, suggesting this protein has an important role in viral RNA replication in virus-infected cells.

Inhibitory effect of serotonin antagonists on JC virus propagation in a carrier culture of human neuroblastoma cells

Human polyomavirus, JCV, causes fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). It has been shown that 5HT2AR acts as a cellular receptor for JCV on human glial cells. In the current study, we examined the inhibitory effects of 5HT2AR antagonists, ketanserin and ritanserin, both on JCV infection and on propagation by using human neuroblastoma cells IMR‐32 and JCI, which continuously produce JCV. Transcriptional analysis revealed that 5HT2AR was constitutively expressed in JCI cells. Treatments with 5HT2AR antagonists led to a significant reduction in the titers of progeny viruses and the population of infected JCI cells. In addition, the amount of JCV genomic DNA was decreased in JCI cells in the presence of 5HT2AR antagonists. These results indicate that 5HT2AR antagonists have an inhibitory effect on JCV infection and reproduction, and JCI cells are applicable to an experimental model for pharmacological evaluation of antiviral agents against JCV.

Scanning electron microscopy with an ionic liquid reveals the loss of mitotic protrusions of cells during the epithelial-mesenchymal transition.

Epithelial–mesenchymal transition (EMT) is a key event in cancer metastasis and is characterized by increase in cell motility, increase in expression of mesenchymal cell markers, loss of proteins from cell‐to‐cell junction complexes, and changes in cell morphology. Here, the morphological effects of a representative EMT inducer, transforming growth factor (TGF)‐β1, were investigated in human lung adenocarcinoma (A549) cells and pancreatic carcinoma (Panc‐1) cells. TGF‐β1 caused morphological changes characteristic of EMT, and immunostaining showed loss of E‐cadherin from cell‐to‐cell junction complexes in addition to the upregulation of the mesenchymal marker vimentin. During scanning electron microscopy (SEM) with an ionic liquid, we observed EMT‐specific morphological changes, including the formation of various cell protrusions. Interestingly, filopodia in mitotic cells were clearly observed by SEM, and the number of these filopodia in TFG‐β1‐treated mitotic cells was reduced significantly. We conclude that this reduction in such mitotic protrusions is a novel effect of TGF‐β1 and may contribute to EMT. Microsc. Res. Tech., 2011.

Inhibitory effect of furanonaphthoquinone derivatives on the replication of Japanese encephalitis virus

Japanese encephalitis still occurs in endemic and epidemic forms over a wide area of Asia. Although the vaccine against Japanese encephalitis virus (JEV) is widely used, no antiviral drug has been reported. We used several different kinds of furanonaphthoquinone derivatives and found antiviral activity against JEV. Especially, 2-methylnaphtho[2,3-b]furan-4,9-dione (FNQ3) indicated the highest antiviral activity, followed by 2-(1-hydroxyethyl)-, 5(or 8)-hydroxy-, and 2-methyl-5(or 8)-hydroxy-analogs of naphtho[2,3-b]furan-4,9-dione. In the presence of 3 microg/ml FNQ3, the virus yields in Vero cells were 2 x 10(5) PFU/ml at 24 h after infecting with the virus and 10% of the control level. Western blot analysis using anti-E rabbit sera or anti-NS3 showed that the expression of viral proteins was inhibited by treatment with FNQ3. In addition, Northern blot analysis indicated that the appearance of JEV-RNA was also inhibited by FNQ3. These results suggest that FNQ3 inhibits JEV replication through viral RNA and protein synthesis.