Abstract-Oxidized phospholipids, including oxidation products of palmitoyl-arachidonyl-phosphatidyl choline (PAPC), are mediators of inflammation in endothelial cells (ECs) and known to induce several chemokines, including interleukin-8 (IL-8). In this study, we show that oxidized PAPC (OxPAPC), which accumulates in atherosclerotic lesions, paradoxically depletes endothelial cholesterol, causing caveolin-1 internalization from the plasma membrane to the endoplasmic reticulum and Golgi, and activates sterol regulatory element-binding protein (SREBP

SummaryCell-cell communication regulates many important processes in bacteria. Gram-positive bacteria use peptide signals for communication, such as the Phr pentapeptides of Bacillus subtilis. The Phr pentapeptides are secreted with a pro domain that is cleaved to produce an active signalling peptide. To identify the protease(s) involved in production of the mature Phr signalling peptides, we developed assays for detecting cleavage of one of the B. subtilis Phr pentapeptides, CSF, from the proCSF precursor. Using both a cellular and a mass spectrometric approach, we determined that a sigma-H-regulated, secreted, serine protease(s) cleaved proCSF to CSF. Mutants lacking the three proteases that fit these criteria, subtilisin, Epr and Vpr, had a defect in CSF production. Purified subtilisin and Vpr were shown to be capable of processing proCSF as well as at least one other Phr peptide produced by B. subtilis, PhrA, but they were not able to process the PhrE signalling peptide of B. subtilis, indicating that there are probably other unidentified proteases involved in Phr peptide production. Subtilisin, Epr and Vpr are members of the subtilisin family of proteases that are widespread in bacteria, suggesting that many bacterial species may be capable of producing Phr signalling peptides.

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Abstract-The lipoxygenase (LO) pathway has been implicated as an important mediator of chronic glucose and platelet-derived growth factor (PDGF)-induced effects in the vascular system. Endothelial cells treated with 12LO products or cultured in high glucose showed enhanced monocyte adhesion, an important step in atherogenesis. We have previously reported that PDGF increased HETE levels in porcine aortic smooth muscle cells. Although several pharmacological inhibitors to the LO pathway are available, most lack specificity and may harbor undesirable side effects. Therefore, we developed a recombinant adenovirus expressing a hammerhead ribozyme (AdRZ) targeted against the porcine leukocyte-type 12LO mRNA to investigate the involvement of LO in glucose-and PDGF-mediated effects in vascular cells. Infection of porcine aortic endothelial cells with AdRZ reduced the level of glucose-enhanced 12LO mRNA expression as determined by quantitative, real-time reverse transcriptase-polymerase chain reaction. Reversephase HPLC and RIA analysis also revealed a corresponding decrease in glucose-stimulated 12HETE production in both the cellular and supernatant fractions. In the ribozyme-treated porcine aortic endothelial cells, there was marked inhibition of high glucose-stimulated monocyte adhesion. Infection with AdRZ also reduced PDGF-induced porcine aortic smooth muscle cell migration by approximately 50%. These studies demonstrate the efficacy of recombinant adenovirus expressing 12LO ribozyme in studying the effects of 12LO in vascular wall cells. Key Words: 12-lipoxygenase Ⅲ ribozyme Ⅲ adenovirus Ⅲ endothelium Ⅲ vascular smooth muscle cells L ipoxygenase (LO) activation has been implicated in numerous diseases including atherosclerosis and diabetes. Certain pathological changes that occur in diabetic vascular disease, such as adhesion of leukocytes to the endothelium and chemotaxis of smooth muscle cells (SMCs), may share a common pathway through LO. Upregulation of LO activity and enhanced formation of LO products have been reported in vitro and in vivo. We and others have reported increased LO activity and elevated levels of LO products, but not cyclooxgenase products, in porcine aortic endothelial cells (PAECs) 1 and porcine SMCs (PSMCs) 2 when cultured in high glucose (HG) conditions. Also, enhanced production of HETEs has been observed in patients with diabetic renal disease 3 and in vessels from infants of diabetic mothers. 4 Exposure to extracellular stimulants such as angiotensin II, platelet-derived growth factor (PDGF), interleukin-4, and interleukin-1 has also led to increased synthesis of LO products in vascular and mononuclear cells. 2,[5][6][7][8] These results suggest that many of the biological effects of the agents involved in stimulating inflammatory processes may be mediated through LO and its products.The LOs responsible for regulating specific vascular functions have not been determined. The number of mammalian LO sequences published includes at least 18 different sequences representing 7 isoforms in 7 ...