The insulin receptor (IR) is expressed ubiquitously in various tissues, where insulin exerts various biological effects on the target cells, such as cellular metabolic changes, cell proliferation and differentiation. Therefore, mimicry of insulin signaling would be a promising strategy to realize artificial control of such cellular fates. In this study, we constructed an antibody/insulin receptor chimera that enables to utilize any antigen as the ligand in principle. We constructed chimeric receptors consisting of antifluorescein single chain Fv (scFv), the extracellular D2 domain of erythropoietin receptor and the transmembrane/intracellular domains of IR (scFv-IR; S-IR). The function of S-IR was evaluated in terms of growth signal transduction in murine pro-B Ba/F3 cells and murine fibroblast NIH/3T3 cells. S-IR exerted IL-3-independent cell growth in Ba/F3 cells, while NIH/ 3T3 cells expressing S-IR acquired growth advantage over parental NIH/3T3 cells in a low-serum condition. S-IR induced phosphorylation of S-IR itself and key signaling molecules downstream of IR. Although antigen-independent activation was significantly observed, S-IR enabled specific amplification of the gene-transduced cells.

The complete nucleotide sequence of the genomic RNA of Tulip virus X Japanese isolate (TVX-J) has been determined. The sequence is 6056 nucleotides in length, excluding the poly(A) tail at the 3' terminus, and contains five open reading frames (ORFs) coding for proteins of Mr 153, 25, 12, 10, and 22 kDa (ORFs 1 through 5, respectively). The genome organization of TVX-J is similar to that of potexviruses, and the encoded proteins share a high degree of homology to the corresponding proteins of other potexviruses. Phylogenetic analyses based on the RNA-dependent RNA polymerase (RdRp) protein (the methyltransferase, helicase, and polymerase domains) encoded by ORF1 and the capsid protein (CP) encoded by ORF5, revealed a close relationship of TVX-J to Plantago asiatica mosaic virus (PlAMV). Pairwise comparison analyses revealed that the relationship between TVX and PlAMV is intermediate between that of strains and species, though previously they have not been considered related. Due to the relatively distant relationships of their replication apparatus and triple gene blocks, we conclude that TVX and PlAMV should be classified as distinct viruses. In addition, the borderline between species and strains of potexviruses is discussed.

The complete nucleotide sequence of the genomic RNA of a Japanese isolate of Potato virus X (PVX), strain 0s (PVX-OS), was determined. The PVX-0s genome is 6435 nucleotides in length, excluding the 3 poly(A) sequence, and encodes for five open reading frames (ORFs), similar to other reported PVX strains. Comparisons of the nucleotide sequence of PVX-0s with those of European and South American PVX strains revealed that the PVX-0s genomic sequence showed 95-96% homology with European strains and 77-78% with South American strains. Phylogenetic analysis of PVX-0s and other PVX strains found that PVX-0s clusters with European strains. This is the first report on the complete nucleotide sequence of a PVX strain from Japan.

As receptor tyrosine kinases (RTKs) play important roles in cell-fate control of various cell types, engineered RTKs that could respond to inexpensive ligands might drastically reduce the cost of producing desired cells for various applications in regenerative medicine. We developed several engineered RTKs named "signalobodies" in which the ligand-recognition domain of RTKs is replaced by single-chain Fv for enabling recognition of a specific antigen. However, the remaining concern was the dysregulation of antigen-dependent on/off signaling of the signalobodies. This study aims at fine-tuning the performance of the signalobodies based on three RTKs (fibroblast growth factor receptor 1, insulin receptor, and c-fms). To this end, the cell-surface expression levels of the RTK-based signalobodies were altered by locating their genes either upstream or downstream of the internal ribosomal entry site, and by inserting 1 to 3 alanine residue(s) at the intracellular juxtamembrane region. As a result, while the signaling response was different among the three signalobodies, the antigen-dependent on/off regulation became tighter when the cell-surface expression levels of the signalobodies were lowered. Therefore, we successfully developed a method to diminish the leaky signaling of RTK-based signalobodies, which will be important for establishing the signalobody-based platform technology that can produce cells of interest for regenerative medicine.

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