Molluscicidal and radical scavenging activity of quinones from the root bark of Caryopteris x clandonensis.

In the search for new molluscicidal natural products, the activity of the chloroform extract of the root barks of Caryopteris x clandonensis was tested. The LC(100) was <5 ppm. The fractionation and purification of the extract afforded 15-deoxyfuerstione, fuerstione and alpha-caryopterone as the main compounds. These compounds were tested against the snail Bulinus truncatus, an intermediate host snail of a schistosomiasis parasite and showed strong molluscicidal activity with LC(100)<4 ppm. In addition, they were found to have potent radical scavenging properties on superoxide radical.

NO-Mediated aromatic nitration during decomposition of phenolic S-nitrosothiols in non-aqueous aerobic medium.

Five novel S-nitrosothiol compounds (6-10) derived from L-cysteine were generated in solution and their decomposition rate was followed by UV spectroscopy. In acetonitrile, compounds 9 and 10 were the most stable of this series with a half-life of 24 h. The final organic decomposition products of the five S-nitrosothiols were also analysed. Derivatives 8, 9, and 10, possessing a phenolic hydroxyl group, afforded an unexpected decomposition pathway, with nitration of aromatic ring occurring in non-aqueous media. A mechanism involving a phenoxy radical seems to be implicated.

Detection of superoxide anion released extracellularly by endothelial cells using cytochrome c reduction, ESR, fluorescence and lucigenin-enhanced chemiluminescence techniques.

Endothelium produces oxygen-derived free radicals (nitric oxide, NO&z.rad;; superoxide anion, O(2)(*-)) which play a major role in physiology and pathology of the vessel wall. However, little is known about endothelium-derived O(2)(*-) production, particularly due to the difficulty in assessing O(2)(*-) when its production is low and to controversies recently raised about the use of lucigenin-enhanced chemiluminescence. We compared four techniques of O(2)(*-) assessment when its production is low. In the present study, we have compared ferricytochrome c reduction, electron spin resonance (ESR) spectroscopy using DMPO as spin trap, hydroethidine fluorescence, and lucigenin-enhanced chemiluminescence to assess O(2)(*-) production in cultured bovine aortic endothelial cells (BAEC). We focused our study on extracellular O(2)(*-) production because the specificity of the signal is provided by the use of superoxide dismutase, and this control cannot be obtained intracellularly. We found that the calcium ionophore A23187 dose-dependently stimulated O(2)(*-) production, with a good correlation between all four techniques. The signals evoked by postconfluent BAEC were increased 2- to 7-fold in comparison to just-confluent BAEC, according to the technique used. Ferricytochrome c 20 microm rather than at 100 microm appears more suitable to detect O(2)(*-). However, in the presence of electron donors such as NADH or NADPH, lucigenin-enhanced chemiluminescence generated high amounts of O(2)(*-). Thus, ferricytochrome c reduction, electron spin resonance (ESR), and hydroethidine fluorescence appear as adequate tools for the detection of extracellular endothelium-derived O(2)(*-) production, whereas lucigenin may be artifactual, even when a low concentration of lucigenin is employed.

An antioxidant sinapic acid ester isolated from Iberis amara.

The isolation of 6-O-sinapoyl sucrose (1) from Iberis amara seeds and an evaluation of its antioxidative properties in comparison with sinapic acid and ascorbic acid are reported.

Novel donors of nitric oxide derived of S-nitrosocysteine possessing antioxidant activities.

Novel S-nitrosothiols possessing a phenolic function were investigated as nitric oxide (NO) donors. A study of NO release from these derivatives was carried out by electron spin resonance (ESR). All compounds gave rise to a characteristic three-line ESR signal in the presence of the complex [Fe(II)(MGD)2], revealing the formation of the complex [Fe(II)(MGD)2(NO)]. Furthermore, tests based on cytochrome c reduction were performed in order to study the ability of each phenolic disulfide, the final organic decomposition product of S-nitrosothiols, to trap superoxide radical anion (O2-). This study revealed a high reactivity of 1b and 3b towards O2-. For these two compounds, the respective inhibitory concentration (IC) 50 values were 92 microM and 43 microM.

Estradiol increases rat aorta endothelium-derived relaxing factor (EDRF) activity without changes in endothelial NO synthase gene expression: possible role of decreased endothelium-derived superoxide anion production.

OBJECTIVES: Estradiol is known to exert a protective effect against atherosclerosis, but the mechanism(s) whereby this protection is mediated is/are unclear. However, estradiol-treated castrated animals exhibit increased activity of endothelium-derived relaxing factor (EDRF), which could contribute to vasculoprotection. In the present work, we investigated the molecular mechanism(s) of the enhancement of EDRF activity in the thoracic aorta of oophorectomized female rats given 17 beta-estradiol (E2, 2 or 40 micrograms/kg/day) compared to those given a placebo. METHODS AND RESULTS: The abundance in the thoracic aorta of NO synthase I, II and III mRNA (using RT-PCR) and of NO synthase I, II and III immunoreactive protein (using Western blotting) was unaltered by E2. NO synthase activity (based on arginine/citrulline conversion) in thoracic aorta homogenates did not differ significantly among the three groups, suggesting that NO production was not enhanced by E2. In contrast, lucigenin-enhanced chemiluminescence of aorta from the E2 group was decreased compared to that of the placebo group. Desendothelialization and exogenously added superoxide dismutase suggested that this difference was due to a decrease in extracellular endothelium-derived production of superoxide anion (O2-.). Experiments in cultured bovine aortic endothelial cells confirmed a decreased extracellular production of O2-. in response to ethinylestradiol (1 nM) using both lucigenin-enhanced chemiluminescence and ESR spectroscopy. Luminol-enhanced chemiluminescence revealed that ethinylestradioltreated cultured endothelial cells generated less peroxynitrite (the byproduct of NO-. and O2-. interaction) than control cells. CONCLUSION: Estradiol increases rat aorta EDRF activity in the absence of changes in endothelial NO synthase gene expression. The decreased endothelium-derived generation of O2-. in response to estrogens could account for enhanced EDRF-NO bioactivity and decreased peroxynitrite release. All of these effects could contribute to the vascular protective properties of estrogens.

2-2'-Pyridylisatogen, a selective allosteric modulator of P2 receptors, is a spin trapping agent.

2-2'-Pyridylisatogen (PIT) has been reported to be a relatively selective irreversible antagonist of responses to adenosine 5'-triphosphate (ATP) in some smooth muscle preparations and to be an allosteric modulator of responses to ATP at recombinant P2Y receptors from chick brain. PIT is also a potent inhibitor of mitochondrial oxidative phosphorylation. However, the compound has a unique nitrone structure, so PIT was compared with dimethyl-pyrroline-N-oxide (DMPO) as a spin trapping agent for superoxide and hydroxyl radicals using electron spin resonance (ESR). PIT was found to be a potent spin trapper of both hydroxyl and superoxide radicals. PIT was more potent than DMPO to trap the hydroxyl radical forming an adduct which was more stable than the DMPO adduct in aqueous media. PIT was an effective spin trap of hydroxyl radical in aqueous buffer at pH 7.4. PIT more slowly trapped the superoxide anion but at concentrations where DMPO trapped none.

Electron spin resonance detection of extracellular superoxide anion released by cultured endothelial cells.

OBJECTIVE AND METHODS: Endothelium produces oxygen-derived free radicals which play a major role in vessel wall physiology and pathology. Whereas NO* production from endothelium has been extensively characterized, little is known about endothelium-derived O2-*. In the present study, we determined the O2-* production of bovine aortic endothelial cells (BAEC) using the spin trap 5,5-dimethyl-1 pyrroline-N-oxide (DMPO) and electron spin resonance (ESR) spectroscopy. RESULTS: An ESR adduct DMPO-OH detected in the supernatant of BAEC after stimulation with the calcium ionophore A23187 originated from the trapping of extracellular O2-*, because coincubation with superoxide dismutase (30 U/ml) completely suppressed the ESR signal, whereas catalase (2000 U/ml) had no effect. A23187 stimulated extracellular O2-* production in a time- and dose-dependent manner. The coenzymes NADH and NADPH both increased the ESR signal, whereas a flavin antagonist, diphenylene iodonium, abolished the ESR signal. Phorbol myristate acetate potentiated, whereas bisindolylmaleimide I inhibited the A23187-stimulated O2-* production, suggesting the involvement of protein kinase C. These signals were not altered L-NAME, a NO-synthase inhibitor, suggesting that the endogenous production of NO* did not alter O2-* production. Finally, the amount of O2-* generated by A23187-stimulated post-confluent BAEC was one order of magnitude higher than that evoked by rat aortic smooth muscle cells stimulated under the same conditions.

Internalization of indium-labeled LDL through a lipid chelating anchor in human pancreatic-cancer cells as a potential radiopharmaceutical for tumor localization.

Low-density lipoproteins (LDL) labeled with indium via a lipid-chelating agent, the bis(stearylamide) of diethylenetri-aminepentaacetic acid (L), were evaluated as a potential radiopharmaceutical (111In-L-LDL) for tumor localization by studying their internalization in human pancreatic cancer cells (Capan-1). Using Dil-LDL (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate-LDL), this cell line was shown to bind human LDL with a high-affinity saturable component and a low-affinity non-saturable (40%) component. The single saturable high-affinity binding site had a KD of 27.5 +/- 2.1 micrograms/ml and a maximal binding of 610 +/- 7.5 ng/ml protein. Electron-microscopic examination of the In-L-LDL particles revealed the peripheral distribution of the electron-dense indium atoms at the outer surface of LDL. The modified LDL were then shown to be internalized by the cells. After conjugation of In-L-LDL to colloidal gold to follow the different stages of internalization, electron-microscopic examination showed that the In-L-LDL gold conjugates were stuck to the external sheet of the plasma apical and microvilli membrane, into earlier and later endosomes and into multivesicular bodies, suggesting the penetration of the In-L-LDL particles into lysosomal vacuoles. The observation of In-L-LDL-gold conjugates in deep-seated cytoplasm suggests that LDL could be employed as a drug-transport vehicle for targeting cytotoxics or radionuclides close to the cell nucleus.

Indium-111 labeling of low density lipoproteins with the DTPA-bis(stearylamide): evaluation as a potential radiopharmaceutical for tumor localization.

In order to use the LDL receptor pathway to target radionuclides to cancer sites for imaging and diagnostic purposes, a labeling procedure of LDL with 111In using the DTPA-bis(stearylamide) (L) has been developed. This bifunctional ligand is intended to be incorporated into the phospholipid monolayer of LDL and to specifically chelate the In3+ cation at the surface. The ligand was incorporated into LDL in buffered medium with a 65-80% yield. The L-LDL samples are stable over a 24 h period when examined by dialysis, allowing their storage before indium-111 radiolabeling. In vitro studies of In-L-LDL particles show that indium labeling is rapidly achieved (1 h). More than 85% of the indium atoms are bound to the chelating functions of the incorporated DTPA derivatives and less than 10% to the nonspecific complexation sites of LDL (e.g., protein residues). After incubation in human serum, the indium activity recovered in the LDL fraction of In-L-LDL samples (95%) is much higher than in In-LDL samples (35%), pointing out the strong stabilizing chelating effect of the ligand. Competitive binding studies show that In-L-LDL are recognized by LDL receptors of A549 cells like native LDL when the In-L/LDL ratio varies from 5 to 30. All these in vitro experiments demonstrate that the In-L-LDL conjugates possess properties suitable for further work with in vivo experiments.

[Thionitrites as potent donors of nitric oxide: example of S-nitroso- and S,S'-dinitroso-dihydrolipoic acids]. / Les thionitries comme agents donneurs potentiels de monoxyde d'azote: exemple des acides S-nitroso- et S,S'-dinitroso-dihydrolipoïque.

Until recently, nitric oxide (NO.) was considered as a toxic radical, but it appears now as an essential messenger implicated in a wide range of biological processes, including immune system, cardiovascular system, and nervous system. An aspect of NO. metabolism in vivo is the formation of a variety of high and low molecular weight nitrosothiols. S-nitrosocysteine and S-nitrosoglutathione are among the biologically derived S-nitrosothiols that are postulated to be carriers of NO.. Although most of the S-nitrosothiols are unstable and spontaneously break down to produce NO. and a disulfide, some of them, including protein thiols, can show significant stability. These molecules are able to convey nitric oxide, that is, to keep, to carry, and then to generate NO. in physiological media, and might display pharmacological effects as potential vasodilators or neuroprotectors. Here, we present the development of new thionitrites R-S-NO having intrinsic antioxidant properties. We report the preparation, the characterization, and the stability studies in aqueous solutions of S-nitroso derivatives of dihydro-alpha-lipoic acid, known for its antioxidant properties.

[Substituted methoxyphenol with antioxidative activity: correlation between physicochemical and biological results]. / Méthoxyphénols substitués à activité antioxydante: corrélation entre les résultats physico-chimiques et biologiques.

Guaiacol moiety has been found in antiinflammatory compounds present in traditional african or chinese medicine. As the activity of these products could be due to reactions with the reactive oxygen species (ROS) or enzymes involved in the inflammatory reaction, a comparative study has been done between biological and physico-chemical investigations. Antioxidant properties of six guaiacol derivatives were measured in vitro by the inhibition of cyclooxygenase activities in human platelets and of the release of ROS by human polymorphonuclear leucocytes (PMNs). PMNs were stimulated by the bacterial peptide N-fMetLeuPhe (FMLP) and the protein kinase C activator phorbol myristate acetate (PMA) using luminol as chemiluminescent probe. Electron Spin Resonance (ESR) and the technique of spin-trapping with 5,5-dimethyl-pyrroline-N-oxide (DMPO) have been used to quantify hydroxyl and superoxide scavenging activities. Hydroxyl radicals were generated by the Fenton's reaction (Fe2+/H2O2) and the superoxide anion by the acetaldehyde/xanthine oxydase system (AC/XOD). The PMNs tests revealed that curcumin and methyl ferulate appeared as the most active compounds. Platelet cycloxygenase activity was inhibited by curcumin and cyclovalone. ESR studies showed a better ROS scavenging activity for vanillin, methyl ferulate and curcumin. Whatever test we used, curcumin and methylferulate appeared as the most interesting antioxidative compounds.

Effect of the amine non-leaving group on the structure and stability of DNA complexes with cis-[Pt(R-NH2)2(NO3)2].

The antitumor compound cis-[Pt(NH3)2Cl2] (cisplatin), conserves two ammine ligands during the reaction with its cellular target DNA. Modifications of these non-leaving groups change the antineoplastic properties of this compound and its genotoxic effects. It is therefore of interest to determine the influence of non-leaving groups on the structure and stability of DNA in vitro. We have investigated platinum-DNA adducts formed by cis-[Pt(R-NH2)2(NO3)2] (where R-NH2 = NH3, methylamine, cyclobutylamine, cyclopentylamine and cyclohexylamine) as a function of DNA binding. All compounds quantitatively reacted with DNA in less than 1 h at 37 degrees C. They formed bifunctional adducts with adjacent nucleotides judging from the displacement of the intercalating molecule ethidium bromide, ultraviolet absorption spectroscopy and circular dichroism. Substitution of a H on the NH3 ligand by alkyl groups dramatically destabilized the platinum-DNA complex. Thermal stability decreased progressively with an increasing number of carbon atoms, delta tm = -4.4 degrees C for 3 cyclohexylamine-platinum-DNA adducts/1000 nucleotides, conditions where cisplatin had no effect. DNA adducts with cyclobutylamine and cyclohexylamine ligands inhibited the hydrolysis of platinum-DNA complexes by S1 nuclease. Km for the digestion of DNA containing these lesions was 2.3 times greater than for cisplatin, indicating steric inhibition of enzyme-substrate complex formation. These results show that the non-leaving groups of substituted cis-Pt(II) compounds may destabilize DNA and interfere with protein-DNA interactions. These perturbations may have consequences for the genotoxic and antitumor activities of platinum compounds.