Cardiotrophin-1 stimulates intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human aortic endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) play critical roles in mediating monocyte adhesion to the vascular endothelium and monocyte migration into the subendothelial regions of the vessels. Inasmuch as cardiotrophin-1 (CT-1), an IL-6-type cytokine, was expressed in human atherosclerotic plaque, we examined whether CT-1 induces monocyte adhesion and migration by stimulating gene and protein expressions of ICAM-1 and MCP-1 in human aortic endothelial cells (HAECs). Immunocytochemistry revealed that CT-1 increased intensity of ICAM-1 and MCP-1 immunoreactivity in HAECs. Adhesion assay and chemotaxis assay revealed that CT-1 increased human monocytic THP-1 cell adhesion to HAECs and promoted chemotaxis in THP-1 cells, which were attenuated by anti-ICAM-1 and anti-MCP-1 antibody, respectively. Western blot analysis showed that CT-1 increased phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt and that their inhibitors, PD-98059, SB-203580, and LY-294002, respectively, inhibited phosphorylation. RNase protection assay and ELISA demonstrated that CT-1 increased gene and protein expressions of ICAM-1 and MCP-1. EMSA revealed that CT-1 enhanced NF-kappaB DNA-binding activity. CT-1-mediated upregulation of ICAM-1 and MCP-1 was suppressed by PD-98059, SB-203580, LY-294002, and parthenolide. The present study demonstrates that CT-1 promotes monocyte adhesion and migration by stimulating ICAM-1 and MCP-1 through mechanisms that involve ERK1/2 MAP kinase, p38 MAP kinase, phosphatidylinositol 3-kinase, and NF-kappaB pathways and suggests that CT-1 plays an important role in the pathophysiology of vascular inflammation and atherosclerosis.     

Characterization of the mac-1 gene encoding a putative ABC transporter from Myxococcus xanthus

The mac-1 gene of Myxococcus xanthus TA, an antibiotic TA producer, encoded a protein with strong sequence similarity to the antibiotic ATP-binding cassette (ABC) transporter for macrolide antibiotics. The mac-1 gene encoding protein (Mac-1) had two ATP-binding domains containing Walker A and B motifs, and no hydrophobic transmembrane regions. Insertional inactivation of mac-1 caused enhanced sensitivity to oleandomycin, a macrolide antibiotic, while the mac-1 mutant showed normal export of antibiotic TA into the extracellular fluid. The mac-1 mutant could form mounds, but was unable to form fruiting bodies or sporulate under nutrient starvation. A primary role for Mac-1 in M. xanthus may be as a transporter which exports or imports a molecule required for the sporulation process.     

Atrial natriuretic peptide (ANP) system in frog skin

In this study the ultrastructure of Rana esculenta skin is described. Cytochemical methods were used to localize guanylate cyclase in the presence of atrial natriuretic peptide and immunocytochemical methods showed the presence of the atrial natriuretic peptide in various levels of skin. The peptide is mainly found in the epithelium and in the lymph sacs of the tela subcutanea. Its receptors are located in the same zones and are indicated by guanylate cyclase activity. We demonstrate that frog skin is a target organ for atrial natriuretic peptide and propose that, at this level, the peptide carries out an important osmoregulatory role.     

Functions of the D-ribosyl moiety and the lower axial ligand of the nucleotide loop of coenzyme B(12) in diol dehydratase and ethanolamine ammonia-lyase reactions

The roles of the D-ribosyl moiety and the bulky axial ligand of the nucleotide loop of adenosylcobalamin in coenzymic function have been investigated using two series of coenzyme analogs bearing various artificial bases. The 2-methylbenzimidazolyl trimethylene analog that exists exclusively in the base-off form was a totally inactive coenzyme for diol dehydratase and served as a competitive inhibitor. The benzimidazolyl trimethylene analog and the benzimidazolylcobamide coenzyme were highly active for diol dehydratase and ethanolamine ammonia-lyase. The imidazolylcobamide coenzyme was 59 and 9% as active as the normal coenzyme for diol dehydratase and ethanolamine ammonia-lyase, respectively. The latter analog served as an effective suicide coenzyme for both enzymes, although the partition ratio (k(cat)/k(inact)) of 630 for ethanolamine ammonia-lyase is much lower than that for diol dehydratase. Suicide inactivation was accompanied by the accumulation of a cob(II)amide species, indicating irreversible cleavage of the coenzyme Co-C bond during the inactivation. It was thus concluded that the bulkiness of a Co-coordinating base of the nucleotide loop is essential for both the initial activity and continuous catalytic turnovers. Since the k(cat)/k(inact) value for the imidazolylcobamide in diol dehydratase was 27-times higher than that for the imidazolyl trimethylene analog, it is clear that the ribosyl moiety protects the reaction intermediates from suicide inactivation. Stopped-flow measurements indicated that the rate of Co-C bond homolysis is essentially unaffected by the bulkiness of the Co-coordinating base for diol dehydratase. Thus, it seems unlikely that the Co-C bond is labilized through a ground state mechanochemical triggering mechanism in diol dehydratase.     

GLC and GLC-mS analysis of thiophene derivatives in plants and in in vitro cultures of Tagetes patula L. (Asteraceae)

The occurrence of thiophenic compounds in diverse plant organs and in in vitro root-, callus- and cell suspension cultures of Tagetes patula cv. Carmen was investigated using capillary GLC and GLC-MS. The separation of thiophenes by capillary GLC and the group specific MS fragmentation with the typical sulfur isotope peaks allowed the unequivocal assignment of individual thiophenes in complex mixtures, even when occurring in traces and in the presence of different geometrical isomers. The extracts of Tagetes patula cv. Carmen contained the following 8 thiophene compounds: 5-(3-buten-1-ynyl)-2,2'-bithienyl (BBT), 5'-methyl-5-(3-buten-1-ynyl)-2,2'-bithienyl (MeBBT), 5-(1-pentynyl)-2,2'-bithienyl (PBT), 5-(4-hydroxy-1-butynyl)-2,2'-bithienyl (BBTOH), 2,2',5,2"-terthienyl (alpha-T), 5-(4-acetoxy-1-butynyl)-2,2'-bithienyl (BBTOAc), 5-methylaceto-5'-(3-buten-1-ynyl)-2,2'-bithienyl (AcOCH2BBT), and 5-(3,4-diacetoxy-1-butynyl)-2,2'-bithienyl (BBT(OAc)2). The most complex thiophene profile, including the less common PBT was detected in aerial parts of freshly harvested plant material. Under in vitro conditions only the root cultures, but not callus or cell suspension cultures produced substantial amounts of irregular thiophenes confirming that roots are the main site of thiophene biosynthesis.