Caveolae targeting and regulation of large conductance Ca(2+)-activated K+ channels in vascular endothelial cells.

The vascular endothelium is richly endowed with caveolae, which are specialized membrane microdomains that facilitate the integration of specific cellular signal transduction processes. We found that the large conductance Ca(2+)-activated K+ (BK) channels are associated with caveolin-1 in bovine aortic endothelial cells (BAECs). OptiPrep gradient cell fractionation demonstrated that BK channels were concentrated in the caveolae-rich fraction in BAECs.

KLF11-mediated repression antagonizes Sp1/sterol-responsive element-binding protein-induced transcriptional activation of caveolin-1 in response to cholesterol signaling.

Cholesterol is a potent regulator of gene expression via a canonical pathway co-regulated by SREBP and Sp1. Here we establish the caveolin-1 gene promoter as a cell type-specific model for SREBP/Sp1 regulation whereby lipoprotein cholesterol depletion activates caveolin-1 transcription in endothelial type cells, but not in fibroblasts, both in vitro and in vivo. By extending this model, we describe a novel pathway distinct from the prototypical SREBP/Sp1 regulatory loop involving the Sp1-like protein, KLF11.

De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase.

Classical swine fever virus nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase, a key enzyme of the viral replication complex. To better understand the initiation of viral RNA synthesis and to establish an in vitro replication system, a recombinant NS5B protein, lacking the C-terminal 24-amino acid hydrophobic domain, was expressed in Escherichia coli. The truncated fusion protein (NS5Bdelta24) was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate either plus- or minus-strand viral RNA synthesis de novo in a primer-independent manner but not by terminal nucleotidyle transferase activity.

Inhibition of GTP-dependent vesicle trafficking impairs internalization of plasmalemmal eNOS and cellular nitric oxide production.

The Ca2+ mobilizing peptide, bradykinin (BK), stimulates endothelial nitric oxide synthase (eNOS)-derived cellular nitric oxide (NO) production in association with altering the subcellular distribution of the enzyme. In the present study we examine the influence of cellular GTPases, particularly the large GTPase dynamin, on BK-mediated eNOS localization and cellular NO production. BK stimulation of ECV cells, which were stably transfected with eNOS-GFP (eNOS-GFP ECV304), increased NO production.

The proline-rich domain of dynamin-2 is responsible for dynamin-dependent in vitro potentiation of endothelial nitric-oxide synthase activity via selective effects on reductase domain function.

The GTPase dynamin-2 (dyn-2) binds and positively regulates the nitric oxide-generating enzyme, endothelial nitric-oxide synthase (eNOS) (Cao, S., Yao, Y., McCabe, T.

Oxidative Modification of Very Low Density Lipoprotein by Arterial Wall Cells.

Modification of VLDL by arterial wall cells was observed. After incubating VLDL (200 &mgr;g protein/ml) with bovine aortic endothelial cells (EC), rabbit aortic smooth muscle cells (SMC) or mouse peritoneal macrophages (Mpsi)for 24 hours, the TBARS in VLDL increased strikingly to 7.80+/-O.