DNA damage-dependent nuclear dynamics of the Mre11 complex.

The Mre11 complex has been implicated in diverse aspects of the cellular response to DNA damage. We used in situ fractionation of human fibroblasts to carry out cytologic analysis of Mre11 complex proteins in the double-strand break (DSB) response. In situ fractionation removes most nucleoplasmic protein, permitting immunofluorescent localization of proteins that become more avidly bound to nuclear structures after induction of DNA damage. We found that a fraction of the Mre11 complex was bound to promyelocyte leukemia protein bodies in undamaged cells. Within 10 min after gamma irradiation, nuclear retention of the Mre11 complex in small granular foci was observed and persisted until 2 h postirradiation. In light of the previous demonstration that the Mre11 complex associated with ionizing radiation (IR)-induced DSBs, we infer that the protein retained under these conditions was associated with DNA damage. We also observed increased retention of Rad51 following IR treatment, although IR induced Rad51 foci were distinct from Mre11 foci. The ATM kinase, which phosphorylates Nbs1 during activation of the S-phase checkpoint, was not required for the Mre11 complex to associate with DNA damage. These data suggest that the functions of the Mre11 complex in the DSB response are implicitly dependent upon its ability to detect DNA damage.

Mre11 complex and DNA replication: linkage to E2F and sites of DNA synthesis.

We show that the Mre11 complex associates with E2F family members via the Nbs1 N terminus. This association and Nbs1 phosphorylation are correlated with S-phase checkpoint proficiency, whereas neither is sufficient individually for checkpoint activation. The Nbs1 E2F interaction occurred near the Epstein-Barr virus origin of replication as well as near a chromosomal replication origin in the c-myc promoter region and was restricted to S-phase cells. The Mre11 complex colocalized with PCNA at replication forks throughout S phase, both prior to and coincident with the appearance of nascent DNA. These data suggest that the Mre11 complex suppresses genomic instability through its influence on both the regulation and progression of DNA replication.

Inhibition of ICE-family cysteine proteases rescues murine lymphocytes from lipoxygenase inhibitor-induced apoptosis.

Two lipophilic derivatives of caffeic acid which inhibit lipoxygenase, caffeic acid phenethyl ester (CAPE) and N,N'-dicyclohexyl-O-(3,4-dihydroxycinnamoyl)-isourea (DCHCU), reduced the proliferative response of murine splenocytes to concanavalin A in vitro. Both CAPE and DCHCU induced apoptosis in murine thymocyte cultures as verified by flow cytometry and by visualisation of DNA with acridine orange staining. CAPE-induced apoptosis was inhibited by z-VAD-fmk, an inhibitor of the interleukin-1beta-converting enzyme family of cysteine proteases. We suggest that the lipoxygenase pathway of arachidonic acid metabolism plays a role in regulating lymphocyte responses such as proliferation and apoptosis.

Competitive inhibition of the 5-lipoxygenase-catalysed linoleate oxidation by arachidonic and 5-hydroperoxy-eicosatetraenoic acids.

Linoleic and arachidonic acids are competing substrates for 5-lipoxygenase from barley. When these two substrates are added simultaneously, arachidonic acid acts as a competitive inhibitor of linoleic acid oxidation with Ki of 20 microM, the same value as the Michaelis constant for arachidonate oxygenation by this enzyme (22 +/- 3 microM). Linoleic acid hydroperoxide accumulated in the reaction mixture does not inhibit the enzymatic process, while arachidonic acid hydroperoxy product (5-hydroperoxy-6,8,11,14-eicosatetraenoic acid) inhibits it with very low Ki equal to 0.5 microM.

Involvement of ecto-ATPase and extracellular ATP in polymorphonuclear granulocyte-endothelial interactions.

The adhesion of human polymorphonuclear granulocytes (PMN) with confluent human endothelial cells (line EAhy926) and with solid substrate coated by collagen and fibronectin (Fn) was studied by phase contrast microscopy and by the measurement of myeloperoxidase activity. The ecto-ATPase inhibitors suramin and Reactive Blue 2 (RB2) more than doubled the adhesion of PMN to endothelial cells. The cells hydrolyzed added ATP and this reaction was inhibited by suramin and RB2. The degree of ATP hydrolysis during PMN adherence depended on solid substrata and decreased in the order: non-stimulated endothelial cells, TNF-stimulated endothelial cells, collagen-coated surface, Fn-coated surface. In the same order adherence increased. The endogenous level of extracellular ATP in the PMN-endothelial coculture was around 25 nM. We conclude that PMN-endothelial adhesion is counteracted by an ecto-ATPase or by ATP receptors with ATPase activity. Such interactions may play a role in PMN rolling and diapedesis as well as in the pathophysiology of PMN activation by an anergic endothelium.

Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties.

Caffeic acid phenethyl ester, an active component of propolis extract, inhibits 5-lipoxygenase in the micromolar concentration range. The inhibition is of an uncompetitive type, i.e. the inhibitor binds to the enzyme-substrate complex but not to the free enzyme. Caffeic acid phenethyl ester also exhibits antioxidant properties. At a concentration of 10 microM, it completely blocks production of reactive oxygen species in human neutrophils and the xanthine/xanthine oxidase system.

The effect of propolis and its components on eicosanoid production during the inflammatory response.

To investigate the possible mechanism of the therapeutic action of propolis, we studied: (a) the effect of propolis, its components, caffeic acid phenethyl ester (CAPE), caffeic acid (CA), quercetin and naringenin, as well as the synthetic compounds indomethacin (IM) and nordihydroguaiaretic acid (NDGA), and a novel lipoxygenase inhibitor N,N'-dicyclohexyl-O-(3,4-dihydroxycinnamoyl)isourea (DCHCU) on eicosanoid production by mouse peritoneal macrophages in vitro; (b) the effect of IM, NDGA, CA, CAPE, DCHCU and propolis on eicosanoid production during acute inflammation in vivo; and (c) the ex vivo and in vivo effect of dietary propolis on arachidonic acid metabolism. The ethanol extract of propolis suppressed prostaglandin and leukotriene generation by murine peritoneal macrophages in vitro and during zymosan-induced acute peritoneal inflammation in vivo. Dietary propolis significantly suppressed the lipoxygenase pathway of arachidonic acid metabolism during inflammation in vivo. CAPE was the most potent modulator of the arachidonic acid cascade among the propolis components examined.

Antimicrobial action of propolis and some of its components: the effects on growth, membrane potential and motility of bacteria.

The effect of the natural bee product propolis on the physiology of microorganisms was investigated using B. subtilis, E. coli and R. sphaeroides. An ethanolic extract of propolis had a bactericidal effect caused by the presence of very active, but labile, ingredients. The exact bactericidal effect of propolis was species dependent: it was effective against gram-positive and some gram-negative bacteria. Propolis and some of its cinnamic and flavonoid components were found to uncouple the energy transducing cytoplasmic membrane and to inhibit bacterial motility. These effects on the bioenergetic status of the membrane may contribute to the antimicrobial action of propolis and its observed synergism with selected antibiotics.

[Lipophilic derivatives of caffeic acid as lipoxygenase inhibitors with antioxidant properties]. / Lipofil'nye proizvodnye kofeinoi kisloty--lipoksigenaznye ingibitory s antioksidantnymi svoistvami.

We have prepared two lipophilic derivatives of caffeic acid at the carboxylic function--caffeic acid phenethyl ester, an active component of propolis, and N,N'-dicyclohexyl-O-(3,4-dihydroxycinnamoyl)-isourea. Both substances inhibit barley 5-lipoxygenase and soybean 15-lipoxygenase at micromolar concentrations. The inhibition is uncompetitive, dose-dependent and reversible. The caffeic acid derivatives also exhibit antioxidant properties and at a concentration 5-10 microM completely block the production of the reactive oxygen species in human neutrophils and in the cell-free xanthine/xanthine oxidase system.

[Lipoxins: study of various biosynthesis pathways]. / Lipoksiny. Issledovanie razlichnykh putei biosinteza.

Lipoxins A4 and B4 were obtained by using soybean lipoxygenase and blood cells as a source of enzymatic activity. The conditions facilitating lipoxin biosynthesis from arachidonic acid catalyzed by soybean 15-lipoxygenase were selected. A comparative analysis of lipoxin biosynthesis with the use of cell suspensions containing only granulocytes and of mixed suspensions (platelets + granulocytes and platelets + total fraction of blood leucocytes) was carried out.