In the work reported by Yokoyama\Kobayashi (1999), this cDNA gene was isolated from a human full\length cDNA bank (Fig

In the work reported by Yokoyama\Kobayashi (1999), this cDNA gene was isolated from a human full\length cDNA bank (Fig.?1b) (GenBank accession no.?”type”:”entrez-nucleotide”,”attrs”:”text”:”AB015628″,”term_id”:”4586833″,”term_text”:”AB015628″AB015628), but its function is unclear. into two types and are capable of functioning as signalling proteins to trigger a series of cellular responses (Kerr & Stark 1991; Malmgaard 2004). Some molecules induced in these responses regulate cell physiological activities and interfere with virus replication (Chesler 2004). Other proteins have functions related to regulation of cell biological activity, for example, nitric oxide synthase (NOS), which can improve the production of NO (Chesler 2004). Recent studies also suggest that IFN\ is capable of inducing expression of many more potent genes in cells (Caraglia 2005). With use of differential display\PCR (DD\PCR) and DNA chip technology, many novel cDNA genes specifically related to IFN\ stimulation have been isolated from cells and examined for biological function (Jurecic & Belmont 2000; Kettunen 2004). Undoubtedly, investigation of such novel genes induced in cellular responses to IFN\ or other IFNs will be NS-1643 useful for detailed study of the molecular mechanisms of IFN\. In the present study, a novel gene has been isolated from human embryo fibroblasts treated with IFN\, and has been found to have the same encoding region as a cDNA gene isolated from a human full\length cDNA bank (Yokoyama\Kobayashi 1999), which encodes a putative type?II membrane protein. This protein, which includes a C\type lectin domain, appears to belong Rabbit Polyclonal to c-Jun (phospho-Tyr170) to the C\type lectin superfamily and probably functions as a receptor interacting with a still\unknown ligand. A typical representative of this superfamily is CD69, an early activating marker in mature T?cells. Although little is known concerning its ligand or mechanism of signal transduction, this protein in the T\cell membrane is probably involved in controlling thymocyte export and the trafficking of T?cells (Weis 1998). In this case, the proteins of the C\type lectin superfamily might be receptor proteins affecting cellular biological properties. Based on a structural comparison of this protein and CD69, it was named type?II membrane protein similar to CD69 (TIIMPSC) (Yokoyama\Kobayashi 1999). The gene encoding it was cloned from human embryo fibroblasts treated with IFN\, and was examined for transcription related to IFN\ stimulation. Further, investigation of the protein coded for by this gene was performed to determine its location, biological effects and signal transduction. The results of this study suggest that NS-1643 expression of this protein, which is up\regulated by IFN\ stimulation, is involved in the control of fibroblast proliferation through the JAK\STAT signalling pathway. MATERIALS AND METHODS Cells NS-1643 Human embryo fibroblast KMB\17 strain cells were used, originated from a primary clone established by the Institute of Medical Biology in 1970; they were maintained in Dulbecco’s modified Eagle’s medium (DMEM)?5% fetal calf serum (FCS) with 1% penicillinCstreptomycin and 2?mm glutamine at 37?C in 5% CO2. Differential display PCR mRNA was harvested from 5??106?KMB\17 cells treated with IFN\ (Sigma, St Louis, MO, USA) at a concentration of 100?/ml for 6?h at 37?C by the QuickPrep micro mRNA purification kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The mRNA of control cells was treated by the same procedure, but without IFN\ treatment. Approximately 0.4?g mRNA re\suspended in 12?l of sterile water was mixed with 2.5?mm oligo(dT) primer and incubated at 70?C for 10?min. After snap cooling on ice, this reaction was continued at 37?C with 10?mm dithiothreitol (Sigma), 20?m dNTP (Sigma) and 200?units of superscript RNaseH?ve reverse transcriptase for 90?min. PCR was performed in a 25\l reaction system containing 1?l cDNA, 1.2?mm MgCl2, 0.05% W\1 detergent, 2?m oligo(dT) primer and 0.5?m random 10mer primers. Cycling parameters were 94?C for 30?s, then 40 cycles of 94?C for 30?s, 40?C for 2?min, and 72?C for 30?s followed by final.