Interactions of imidazoline ligands with the active site of purified monoamine oxidase A

The two forms of monoamine oxidase, monoamine oxidase A and monoamine oxidase B, have been associated with imidazoline-binding sites (type 2). Imidazoline ligands saturate the imidazoline-binding sites at nanomolar concentrations, but inhibit monoamine oxidase activity only at micromolar concentrations, suggesting two different binding sites [Ozaita A, Olmos G, Boronat MA, Lizcano JM, Unzeta M & García-Sevilla JA (1997) Br J Pharmacol121, 901-912]. When purified human monoamine oxidase A was used to examine the interaction with the active site, inhibition by guanabenz, 2-(2-benzofuranyl)-2-imidazoline and idazoxan was competitive with kynuramine as substrate, giving K(i) values of 3 microM, 26 microM and 125 microM, respectively. Titration of monoamine oxidase A with imidazoline ligands induced spectral changes that were used to measure the binding affinities for guanabenz (19.3 +/- 3.9 microM) and 2-(2-benzofuranyl)-2-imidazoline (49 +/- 8 microM). Only one type of binding site was detected. Agmatine, a putative endogenous ligand for some imidazoline sites, reduced monoamine oxidase A under anaerobic conditions, indicating that it binds close to the flavin in the active site. Flexible docking studies revealed multiple orientations within the large active site, including orientations close to the flavin that would allow oxidation of agmatine.     

Salix kaptarae sp. nov. (Salicaceae) from Crete

A new species of willow from Crete, named Salix kaptarae Cambria, C. Brullo & Brullo, is described and illustrated. Morphologically, it shows close relationships with species belonging to the S. apennina group, mainly with respect to the leaf shape, and the hairy ovary, capsule and base of stamina filaments. Considerations on its ecology, leaf micro‐morphology and conservation status are also provided.     

Pyrite and Organic Compounds Coexisting in Intrusive Mafic Xenoliths (Hyblean Plateau,Sicily): Implications for Subsurface Abiogenesis

Origins of Life and Evolution of Biospheres - Pyrite and organic matter closely coexist in some hydrothermally-altered gabbroic xenoliths from the Hyblean Plateau, Sicily. The representative sample...     

Ligation of the α2-macroglobulin signaling receptor on macrophages induces synthesis of platelet activating factor

The binding of receptor-recognized forms of α₂-macroglobulin (α₂M) to macrophage α₂M signaling receptors increases inositol-1,4,5-triphosphate synthesis and induces Ca²⁺ mobilization. In this report, we demonstrate that ligation of the macrophage α₂M signaling receptor is also associated with synthesis of platelet activating factor (PAF) by both the de novo and remodeling pathways. Both α₂M-methylamine and a cloned and expressed 20-kDa receptor binding fragment (RBF) from rat α₂M+, stimulated macrophage synthesis of PAF from [³H]acetate, [³H]methylcholine, and 1-O-[³H]alkyl lyso-PAF by two- to threefold. PAF levels reached a peak in 20 min after the cells were exposed to α₂M-methylamine or RBF; they remained elevated for about 1 h after ligand addition to the cells. When [³H]methylcholine was the substrate, pertussis toxin did not block PAF synthesis, but the protein kinase C inhibitor staurosporin reduced synthesis by 65–70%. Cycloheximide completely abolished the increase in synthesis of PAF by macrophages exposed to α₂M-methylamine. By contrast, when [³H]acetate was employed as a precursor, staurosporin or cycloheximide did not abolish the increase in PAF synthesis. These studies suggest that protein kinase C is necessary for the induction of the de novo pathway by α₂M-methylamine. Both α₂M-methylamine and RBF stimulated the activity of lyso-PAF acetyltransferase by about fourfold. Both ligands also stimulated the activity of PAF acetylhydrolase by about six- to sevenfold, indicating that ligation of the α₂M signaling receptor also regulates the degradation of PAF. The ability of receptor-recognized forms of α₂M to regulate levels of PAF suggests that α₂M-proteinase complexes not only regulate macrophage function by activating intracellular signaling but also may indirectly regulate the function of other cells that cannot bind α₂M-proteinase complexes. © 1996 Wiley-Liss, Inc.     

G Protein β Subunit–null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein β subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type β subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In β subunit–null cells, and in a series of β subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein–actin transfected cells showed that β subunit– null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca²⁺ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.     

Assessing the role of the T cell receptor beta gene enhancer in regulating coding joint formation during V(D)J recombination

To assess the role of the T cell receptor (TCR) beta gene enhancer (Ebeta) in regulating the processing of VDJ recombinase-generated coding ends, we assayed TCRbeta rearrangement of Ebeta-deleted (DeltaEbeta) thymocytes in which cell death is inhibited via expression of a Bcl-2 transgene. Compared with DeltaEbeta, DeltaEbeta Bcl-2 thymocytes show a small accumulation of TCRbeta standard recombination products, including coding ends, that involves the proximal Dbeta-Jbeta and Vbeta14 loci but not the distal 5' Vbeta genes. These effects are detectable in double negative pro-T cells, predominate in double positive pre-T cells, and correlate with regional changes in chromosomal structure during double negative-to-double positive differentiation. We propose that Ebeta, by driving long range nucleoprotein interactions and the control of locus expression and chromatin structure, indirectly contributes to the stabilization of coding ends within the recombination processing complexes. The results also illustrate Ebeta-dependent and -independent changes in chromosomal structure, suggesting distinct modes of regulation of TCRbeta allelic exclusion depending on the position within the locus.     

Diterpenoid resin acids of Daemonorops draco

Chromatography of a cyclohexane extract of commercial “dragon's blood” resin yielded a fraction containing pimaric, isopimaric, dehydroabietic and abietic acids. A fifth component of the mixture was tentatively identified as sandaracopimaric acid.     

6-Mer hyaluronan oligosaccharides increase IL-18 and IL-33 production in mouse synovial fibroblasts subjected to collagen-induced arthritis

Hyaluronan (HA) oligosaccharides stimulate pro-inflammatory responses in different cell types by modulating both cluster determinant 44 (CD44) and TLR4. The activation of these receptors is also mediated by collagen-induced arthritis (CIA) that, via two different pathways, culminates in the liberation of NF-κB. This then stimulates the production of pro-inflammatory cytokines, including IL-18 and IL-33, that are greatly involved in rheumatoid arthritis. The aim of this study was to investigate the effects of 6-mer HA oligosaccharides on mouse synovial fibroblasts obtained from normal DBA/J1 mice or mice subjected to CIA. Compared with normal synovial fibroblasts (NSF), rheumatoid arthritis synovial fibroblasts (RASF) showed no up-regulation of CD44 and TLR4 mRNA expression and the related proteins, as well as no activation of NF-κB. Very low levels of both mRNA and related proteins were also detected for IL-18 and IL-33. Treatment of NSF and RASF with 6-mer HA oligosaccharides significantly increased all the parameters in both fibroblast groups, although to a greater extent in RASF. The addition of hyaluronan binding protein to both NSF and RASF inhibited HA activity and was able to reduce the effects of 6-mer HA oligosaccharides and the consequent inflammatory response.     

Tuberculosis unleashed: the impact of human immunodeficiency virus infection on the host granulomatous response to Mycobacterium tuberculosis

The granuloma plays a critical role in the host immune response to Mycobacterium tuberculosis, containing the organism and confining it in a latent state in most infected individuals. Indeed, approximately one-third of the world's population has latent M. tuberculosis infection. However, over the past decade, the human immunodeficiency virus type 1 (HIV-1) pandemic has profoundly affected the incidence and clinicopathological features of tuberculosis. This review examines the immunological mechanisms whereby HIV-1 impairs the establishment, maintenance and function of the tuberculous granuloma.