We show here that the immediate upstream region (from position -12 to -270) of the murine interleukin 4 (Il-4) gene harbors a strong cell-type specific transcriptional enhancer. In T lymphoma cells, the activity of the Il-4 promoter/enhancer is stimulated by phorbol esters, Ca++ ionophores and agonists of protein kinase A and inhibited by low doses of the immunosuppressant cyclosporin A. The Il-4 promoter/enhancer is transcriptionally inactive in B lymphoma cells and HeLa cells. DNase I footprint protection experiments revealed six sites of the Il-4 promoter/enhancer to be bound by nuclear proteins from lymphoid and myeloid cells. Among them are four purine boxes which have been described to be important sequence motifs of the Il-2 promoter. They contain the motif GGAAA and are recognized by the inducible and cyclosporin A-sensitive transcription factor NFAT-1. Three of the Il-4 NFAT-1 sites are closely linked to weak binding sites of Octamer factors. Several purine boxes and an AT-rich protein-binding site of the Il-4 promoter are also recognized by the high mobility group protein HMG I(Y). Whereas the binding of NFAT-1 and Octamer factors enhance the activity of the Il-4 promoter, the binding of HMG I(Y) suppresses its activity and, therefore, appears to be involved in the suppression of Il-4 transcription in resting T lymphocytes.

The onset of DNA replication is an important step within the life cycle of the human neurotropic polyomavirus JC. In this report, evidence that both the human and the murine tumor suppressor protein p53 strongly inhibit JCV DNA replication in vivo is presented. This inhibition is dose-dependent and not a secondary effect of a decreased expression of JCV large T-antigen in response to p53. Using deletion mutants of murine p53 and tumor-derived point mutations of human p53, the basis of the suppression of JCV DNA replication by p53 was dissected. Deletion of either the amino- or the carboxy-terminal domain of murine p53 did not interfere with the repression of JCV DNA replication. However, deletion of the highly conserved central region of p53 abolished the inhibitory effect on replication. The tumor-derived human mutant p53(His273) inhibited JCV DNA replication significantly, whereas another tumorigenic mutant, p53(His175), had no inhibitory effect Concomitantly, a direct protein-protein interaction between p53 and JCV large T-antigen was lost in mutants which did not affect JCV DNA replication. These results strongly suggest that p53 inhibits JCV DNA replication by interacting with JCV large T-antigen.

Pathological nitric oxide (NO) generation in sepsis, inflammation, and stroke may be therapeutically controlled by inhibiting NO synthases (NOS). Here we targeted the (6R)-5,6,7,8-tetrahydro-L-biopterin (H 4 Bip)-binding site of NOS, which, upon cofactor binding, maximally increases enzyme activity and NO production from substrate L-arginine. The first generation of H 4 Bip-based NOS inhibitors employed a 4-amino pharmacophore of H 4 Bip analogous to antifolates such as methotrexate. We developed a novel series of 4-oxo-pteridine derivatives that were screened for inhibition against neuronal NOS (NOS-I) and a structure-activity relationship was determined. To understand the structural basis for pterin antagonism, selected derivatives were docked into the NOS pterin binding cavity. Using a reduced 4-oxo-pteridine scaffold, derivatives with certain modifications such as electron-rich aromatic phenyl or benzoyl groups at the 5-and 6-positions, were discovered to markedly inhibit NOS-I, possibly due to hydrophobic and electrostatic interactions with Phe 462 and Ser 104 , respectively, within the pterin binding pocket. One of the most effective 4-oxo compounds and, for comparisons an active 4-amino derivative, were then co-crystallized with the endothelial NOS (NOS-III) oxygenase domain and this structure solved to confirm the hypothetical binding modes. Collectively, these findings suggest (i) that, unlike the antifolate principle, the 4-amino substituent is not essential for developing pterinbased NOS inhibitors and (ii), provide a steric and electrostatic basis for their rational design.

MuNTS2, a 423 bp sequence isolated from the non-transcribed spacer of murine rDNA stimulates the amplification of cis-linked plasmid DNA in mouse cells under selective conditions. Here we demonstrate that a 180 bp subdomain of muNTS2 is highly homologous (approximately 70%) to three domains of the first well-characterized origin of replication of mammalian chromosomes, i.e. the origin of bidirectional replication (OBR) of the dihydrofolate reductase (DHFR) locus in Chinese hamster ovary (CHO) cells. When subcloned, the 180 bp homology region of muNTS2 was revealed to be essential for the amplification promoting activity of muNTS2. Fragments of the initiation zone of DNA replication from the DHFR locus of hamster cells containing the domains of homology to the mouse muNTS2 element proved also to promote DNA amplification. Thus, the screening system for amplification promoting elements turned out to detect an origin of bidirectional replication.

MuNTS2, a 423 bp sequence isolated from the non-transcribed spacer of murine rDNA stimulates the amplification of cis-linked plasmid DNA in mouse cells under selective conditions. Here we demonstrate that a 180 bp subdomain of muNTS2 is highly homologous (approximately 70%) to three domains of the first well-characterized origin of replication of mammalian chromosomes, i.e. the origin of bidirectional replication (OBR) of the dihydrofolate reductase (DHFR) locus in Chinese hamster ovary (CHO) cells. When subcloned, the 180 bp homology region of muNTS2 was revealed to be essential for the amplification promoting activity of muNTS2. Fragments of the initiation zone of DNA replication from the DHFR locus of hamster cells containing the domains of homology to the mouse muNTS2 element proved also to promote DNA amplification. Thus, the screening system for amplification promoting elements turned out to detect an origin of bidirectional replication.