In this study, a B cell growth stimulatory factor, constitutively secreted by a human CD4+ T cell hybridoma clone, MP6, has been purified and characterized. Serum-free 24 h culture media from MP6 cells were collected, concentrated by ultrafiltration and separated by gel chromatography. Fractions were analyzed for stimulatory activity using [3H]thymidine incorporation in normal and leukemic (B-CLL) B cells as target cells. Activity was present in a 12 kDa protein peak. Upon storage this lost activity indicating that the factor was sensitive to air oxidation, a well-known property of mammalian thioredoxins (Trxs). Treatment of the inactive fraction with dithiothreitol restored full activity. When culture medium was analyzed with a radioimmunoassay for human placenta Trx, the MP6 clone was shown to release 30-50 ng/ml per million cells during 24 h. The B cell stimulatory activity of the MP6 medium was removed by Sepharose-bound anti-human placenta Trx IgG and activity was recovered by elution from the antibodies. Furthermore, MP6 medium showed Trx activity with NADPH and Trx reductase using an insulin disulfide reduction assay. Starting from 5 l of serum-free MP6 conditioned medium, the Trx was purified approximately 100,000-fold. After gel electrophoresis banding, the material was analyzed by peptide sequencing and a full length sequence of an 104 amino acid long protein was obtained. This Trx sequence was identical to the previously published sequence of human Trx from HTLV-1 transformed T cells, adult T cell leukemia-derived factor/Trx.(ABSTRACT TRUNCATED AT 250 WORDS)

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C-CAM is a Ca2+-independent rat cell adhesion molecule belonging to the CEA gene family of the immunoglobulin superfamily. Two major isoforms that differ in the length of their cytoplasmic domains exist. In previous studies it has been reported that only the long isoform (C-CAM1) but not the short isoform (C-CAM2) can mediate adhesion. However, in the mouse, isoforms with both long and short cytoplasmic domains have been reported to have adhesive activity. In order to analyze this apparent conflict we transfected C-CAMI or C-CAM2 into CHO Pro5 cells and examined their adhesive phenotype in an aggregation assay. We found that in this cellular system both C-CAMI and C-CAM2 could mediate cell-cell adhesion in a Ca2+-independent and temperature-independent way. The results suggest that the cellular environment is important for the activity of C-CAM isoforms.

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is the only one of the EBNA proteins to have been implicated as an EBV-encoded transforming protein. More detailed studies of this protein have been hampered by the lack of EBNA-2-specific monoclonal antibodies (MAbs) and of purified protein. To overcome these problems, we isolated 5 hybridomas producing MAbs reactive with an 18 residue synthetic peptide corresponding to the carboxyterminus of EBNA-2. Four of the 5 MAbs were specifically reactive with EBNA-2 in its denatured form on immunoblots. The 5th antibody (115E) was reactive with the native form of EBNA-2. By using a one-step immunoaffinity purification method with 115E cross-linked to protein-A-Sepharose, we purified EBNA-2 to homogeneity, i.e., more than 1,200-fold, from Burkitt lymphoma cell extracts. A major 32-kDa associated protein and a less abundant 17-kDa protein were co-purified with EBNA-2. Immunoprecipitation with 115E from 35S-methionine-labelled cell extracts showed that the 32-kDa protein co-precipitated with EBNA-2 from EBV-positive cells, but was not detectable in immunoprecipitates of EBV-negative cells. When the immunoprecipitates or the purified proteins were immunoblotted with EBV-immune sera, only EBNA-2 was reactive, indicating that the associated proteins are of cellular origin. Immunoprecipitation of cells labelled with 32P-orthophosphate showed that EBNA-2, but not the associated proteins, is a phosphoprotein. The expression level of EBNA-2 varied between different EBV-carrying cell lines, as measured by a 2-site ELISA based on antibody 115E. In indirect immunofluorescence, the 115E MAb gave an EBNA-2-specific characteristic granular staining pattern. These characteristics of EBNA-2 resemble those of other viral transforming proteins.