No abstract

No abstract

A full-length cDNA, encoding the mouse atrial natriuretic peptide clearance receptor (ANP-CR), was isolated from a mouse lung cDNA library. The deduced amino acid sequence of the mouse ANP-CR, showing a typical tripartite organization which lacks a guanylyl cyclase domain, was extremely well conserved compared with the ANP-CR homologs. To understand the molecular mechanisms underlying the regulation of mouse ANP-CR gene expression and to define the essential DNA sequences for the transcriptional activity, a genomic clone containing over 9 kb of the 5'-flanking region of the mouse ANP-CR gene has been isolated from a mouse genomic library. Sequence analysis revealed that the 2.3-kb region upstream from an ATG codon of the mouse ANP-CR gene contained a number of putative regulatory elements; TATA box, CAAT box, CAMP response element, AP-I and two shear stress responsive elements. Additionally, an unusual feature was the presence of the tandem-repeated AP-2-like elements, which were closely overlapped with SP-1 element. Promoter analysis using deletion plasmids in mouse Balb/3T3 cells, highly producing ANP-CR mRNA, demonstrated that deletion of the sequence from -144 to +46 relative to the transcription start point caused a dramatic decrease of the transcriptional activity and that the TATA box at -269 was not essential for the basal transcriptional activity. Primer extension analysis indicated that transcription of the mouse ANP-CR gene starts from at least two major sites, suggesting that the sequence from -144 to +46, which was shown to involve a novel sequence composed of tandem-repeated TATA-box-like elements, contained promoter sequences. Furthermore, cisacting negative elements were shown to be situated in three regions (from -1178 to -708, from -707 to -625 and from -248 to -145) of the mouse ANP-CR gene promoter.Keywords: mouse; natriuretic peptide; receptor; cDNA; genome.The atrial natriuretic peptide family is now recognized to be composed of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) [l, 21. ANP and BNP have been considered to play important roles in the control of systemic blood pressure and intravascular volume [3]. Atrial natriuretic peptide clearance receptor (ANP-CR) which has a very short putative intracytoplasmic extension with no guanylyl cyclase activity [4-61 is thought to regulate the biological effects of natriuretic peptides by eliminating the peptides from the blood circulation. In addition, ANP-CR has been demonstrated to mediate adenylyl cyclase activity via Gi protein, phospholipid hydrosis and thymidine kinase activity [7 -91 without any cyclic cGMP response. ANP-CR gene expression has been reported to be regulated by ANP and endothelium-derived relaxing factor (EDRF) [lo], which are key mediators for protection of cardiovascular diseases, and jl-2-adrenergic stimulants Abbreviurions. ANP, atrial natriuretic peptide; ANP-CR, atrial natriuretic peptide clearance receptor; BAEC, bovine aortic endothelial cell ; VSMC, vascular sm...

The C-terminal two-thirds of nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses RNA helicase activity. This enzyme is considered to be involved in viral replication, and is expected to be one of the target molecules of anti-HCV drugs. Previously, we established a high-throughput screening system for HCV helicase inhibitors using the Scintillation Proximity Assay (SPA) system [Kyono, K. et al. (1998) ANAL: BIOCHEM: 257, 120-126]. Here, we show improvement of the preparation method for the HCV NS3/4A complex. Alteration of the expression region led to an increase in protein expression. The partially purified full-length NS3 protein showed higher NS3 protease activity without the cofactor NS4A peptide than the truncated protease domain with the cofactor peptide, suggesting that this protein formed a complex with NS4A. NS3 further purified to homogeneity, as judged on silver staining, remained in a complex with NS4A. Characterization of the helicase activity of this full NS3/4A complex using the SPA helicase assay system revealed that this enzyme preferred Mn(2+), and that the optimal pH was 6.0-6.5. The NS3/4A complex could act on a DNA template but could not unwind the M13DNA/DNA substrate.

2,2'-Disubstituted biphenyl compounds (1-15) were synthesized by using the Ullmann coupling reaction as a key step. The suppressive effect of these compounds against CCl4-induced liver injuries in mice was evaluated. An unsymmetrical biphenyl (14f) exhibited the most potent activity. The structure-activity relationship is discussed.