We have conducted an in silico data base search for and cloned a novel G-protein-coupled receptor (GPCR) named TG1019. Dot and Northern blotting analyses showed that transcripts of the novel GPCR were expressed in various tissues except brain, and the expression was more intense in liver, kidney, peripheral leukocyte, lung, and spleen than in other tissues. By GTP␥S binding assay using the TG1019-G␣ i1 -protein fusion expressed in insect cells, eicosanoids, and polyunsaturated fatty acids such as 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE), 5(S)-hydroperoxy-6E,8Z, 11Z,14Z-eicosatetraenoic acid, and arachidonic acid were identified to exhibit agonistic activities against TG1019. 5-oxo-ETE was the most potent to enhance the specific binding by 6-fold at a maximum effect dose of submicromolar to micromolar order with an ED 50 value of 5.7 nM. Conversely, polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid showed antagonistic activities against TG1019. In Chinese hamster ovary cells transiently expressing TG1019, the forskolin-stimulated production of cAMP was inhibited up to ϳ70% by 5-oxo-ETE, with an IC 50 value of 33 nM. This inhibition was sensitive to pretreatment of the cells with pertussis toxin.

The level of fibronectin (FN) gene transcription in resting rat 3Y1 cells is very high but decreases steeply after growth stimulation by serum or by the induction of EIA expression. To study the mechanism of this ElA-mediated down-regulation, the 5' flanking regions of the FN gene with various deletions and substitutions were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and introduced into resting 3Y1 cells with ElA expression plasmids. The results indicate that the G10 stretch located from nucleotide position -239 to -230 and two GC boxes from position -105 to -95 and position -54 to -44 are the primary ElA-responsive elements for repression of the FN gene. Two GC boxes also contain a GIO stretch that is interrupted by the presence of an internal C residue. These sequences overlap with the Spl motif GGGCGG. The ElA gene of human adenovirus type 2 (Ad2) and adenovirus type 5 generates two major species of mRNAs with sizes of 13 and 12S that encode proteins of 289 and 243 amino acid residues (289R and 243R). These proteins have identical N-and C-terminal sequences but differ by the presence of an internal 46-amino-acid sequence in the 289R protein. The domain for transactivation of a variety of viral and cellular promoters resides in this internal sequence specific to the 289R protein (2), and the domain for repression of the enhancer-dependent promoters (4,18,46) resides in the first exon common to these proteins. The ElA gene has the ability to transform primary rodent cells in collaboration with a second oncogene (38,53). This transforming function may be closely related to its functions for immortalizing primary cells (11, 23) and inducing cellular DNA synthesis in quiescent cells (1,28,36,43 ElA expression (20). This is in contrast to mouse embryo-derived AKR-2B cells (3), NIH 3T3 cells (40), and the human fibrosarcoma cell line HT-1080 (13), in which expression levels of FN, its receptor, and actin increased coordinately shortly after growth stimulation of quiescent cells by serum. FN is a large glycoprotein of the extracellular matrix which plays a central role in cell anchorage through cell-extracellular matrix interaction (24, 39, 51) and connects intracellular actin filaments to the extracellular matrix by binding to its receptor (7,25). The fact that FN expression is required for cell proliferation is somewhat paradoxical. Cell adhesion to the substratum seems to be required in order for quiescent cells to exit from the Go phase, while the loosening of cell adhesion is required for cell cycle progression transiently stimulated by extracellular proteases (8,45) or by growth factors (21,22). The latter process is mediated by the structural modification of focal contacts via intracellular mediators. The level of FN expression is greatly reduced in v-src-transformed cells (15) and in primary rat kidney cells transformed by the EJ Ha-ras and the ElA genes (26).In the present study, the ElA-responsive elements for repression of FN gene transcription in rat 3Y1 cells were invest...

Downregulation of the fibronectin (FN) gene in a rat 3Y1 derivative cell line, XhoC, transformed by the adenovirus E1A and E1B genes seems to be caused by the induction of a negative regulator, G10BP, which binds to three G-rich sequences in the promoter (T. Nakamura, T. Nakajima, S. Tsunoda, S. Nakada, K. Oda, H. Tsurui, and A. Wada, J. Virol. 66:6436-6450, 1992). These are the G 10 stretch and two GC boxes consisting of the G 10 stretch with one internal C residue insertion. The recognition sequences of G10BP and Sp1 (GGGCGG) overlap in these GC boxes. To analyze the mechanism of the downregulation, G10BP was purified by DNA affinity chromatography, and its molecular mass was estimated to be about 30 kDa. The promoter was modified by substituting the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, and by replacing the Sp1 motif by the T stretch. Transcription of FN promoter-chloramphenicol acetyltransferase fusion genes carrying the base substitution in one or more of these G-rich sequences both in vivo and in vitro revealed that the base substitution in any G-rich sequence results in reduction of promoter activity, although the downstream GC box (GCd) plays a primary role. The addition of G10BP severely inhibited the activities of the FN promoters carrying the wild-type GCd in vitro, while the promoters carrying the mutant GCd were unaffected. The binding affinity of G10BP and Sp1 to each of the G-rich sequences, analyzed by gel shift assays, indicated that G10BP binds strongly to the GCd, moderately to the G 10 stretch, and weakly to GCu, while Sp1 binds strongly to GCu, moderately to GCd, and weakly to the G 10 stretch. Sp1 binding to GCd and the G 10 stretch was inhibited by G10BP, while binding to GCu was unaffected. These results indicate that FN gene transcription is inhibited in XhoC cells primarily by exclusion of Sp1 binding to GCd by G10BP and that G10BP is a new class of Sp1 negative regulator.Fibronectins (FNs) are large glycoproteins of the extracellular matrix and play a central role in cell adhesion, migration, wound healing, and tumor metastasis (24). FN binds to its receptor on the cell surface as dimers of similar subunits of about 250 kDa (24, 44), connecting intracellular actin filaments to the extracellular matrix at the receptor site (2, 23, 44), called focal contacts or adhesion plaques. The FN molecule consists of a series of globular domains which interact independently with components of the extracellular matrix, fibrin, glycosaminoglycans, collagen, and its receptor (22,50), and the receptor binds indirectly to actin filaments through its cytoplasmic domain (4, 21).Cell adhesion to the substratum is required for mammalian cells to proliferate in an in vitro culture system, while the loosening of cell adhesion is also required transiently for quiescent cells to enter the cell cycle after growth stimulation by extracellular proteases (3, 45) or by growth factors (19,20). Most oncogenes exert their functions to disintegrate the structure of the ...

A new antibiotic designated TMC-69has been isolated from,the fermentation broth of a fungal strain Chrysosporium sp. TC 1068. TMC-69exhibited moderate in vitro cytotoxic activity. TMC-69-6H, a derivative of TMC-69prepared by hydrogenation, possessed more potent in vitro cytotoxicity than TMC-69, and exhibited in vivo antitumor activity against murine P388 leukemia and B16 melanoma. TMC-69-6H was found to specifically inhibit Cdc25Aand B phosphatases.

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