Myricitrin as a substrate and inhibitor of myeloperoxidase: implications for the pharmacological effects of flavonoids

Flavonoids are increasingly being ingested by the general population as chemotherapeutic and anti-inflammatory agents. They are potentially toxic because of their conversion to free radicals and reactive quinones by peroxidases. Little detailed information is available on how flavonoids interact with myeloperoxidase, which is the predominant peroxidase present at sites of inflammation. This enzyme uses hydrogen peroxide to oxidize chloride to hypochlorous acid, as well as to produce an array of reactive free radicals from organic substrates. We investigated how the flavonoid myricitrin is oxidized by myeloperoxidase and how it affects the activities of this enzyme. Myricitrin was readily oxidized by myeloperoxidase in the presence of hydrogen peroxide. Its main oxidation product was a dimer that underwent further oxidation. In the presence of glutathione, myricitrin was oxidized to a hydroquinone that was conjugated to glutathione. When myeloperoxidase oxidized myricitrin and related flavonoids it became irreversibly inactivated. The number of hydroxyl groups in the B ring of the flavonoids and the presence of a free hydroxyl m-phenol group in the A ring were important for the inhibitory effects. Less enzyme inactivation occurred in the presence of chloride. Neutrophils also oxidized myricitrin to dimers in a reaction that was partially dependent on myeloperoxidase. Myricitrin did not affect the production of hypochlorous acid by neutrophils. We conclude that myricitrin will be oxidized by neutrophils at sites of inflammation to produce reactive free radicals and quinones. It is unlikely to affect hypochlorous acid production by neutrophils.     

In vivo antimalarial activity of novel 2-hydroxy-3-anilino-1,4-naphthoquinones obtained by epoxide ring-opening reaction

1,4-Naphthoquinone derivatives are known to have relevant activities against several parasites. Among the treatment options for malaria, atovaquone, a 1,4-naphthoquinone derivative, is widely applied in the treatment and prophylaxis of such disease. Based on the structure simplification of atovaquone, we designed and synthesized four novel naphthoquinoidal derivatives. The compounds were obtained by the underexplored epoxide-opening reaction of 1,4-naphthoquinone using aniline derivatives as nucleophiles. The antiplasmodial activity of the synthesized compounds was performed in vivo using Peter’s 4 days suppression test. Significant parasitemia reduction and increased survival were observed for some of the compounds.     

An arachidonic acid generation/export system involved in the regulation of cholesterol transport in mitochondria of steroidogenic cells

Recent studies demonstrated the importance of the mitochondrial ATP in the regulation of a novel long-chain fatty acid generation/export system in mitochondria of diabetic rat heart. In steroidogenic systems, mitochondrial ATP and intramitochondrial arachidonic acid (AA) generation are important for steroidogenesis. Here, we report that mitochondrial ATP is necessary for the generation and export of AA, steroid production and steroidogenic acute regulatory protein induction supported by cyclic 3'-5'-adenosine monophosphate in steroidogenic cells. These results demonstrate that ATP depletion affects AA export and provide new evidence of the existence of the fatty acid generation and export system involved in mitochondrial cholesterol transport.     

Connectivity in the network macrostructure of Tursiops truncatus in the Pelagos Sanctuary (NW Mediterranean Sea): does landscape matter?

The bottlenose dolphin (Tursiops truncatus Montagu, 1821) is a regularly observed species in the Mediterranean Sea, but its network organization has never been investigated on a large scale. We described the network macrostructure of the bottlenose dolphin (meta)population inhabiting the Pelagos Sanctuary (a wide protected area located in the north-western portion of the Mediterranean basin) and we analysed its connectivity in relation to the landscape traits. We pooled effort and sighting data collected by 13 different research institutions operating within the Pelagos Sanctuary from 1994 to 2011 to examine the distribution of bottlenose dolphins in the Pelagos study area and then we applied a social network analysis, investigating the association patterns of the photo-identified dolphins (806 individuals in 605 sightings). The bottlenose dolphin (meta)population inhabiting the Pelagos Sanctuary is clustered in discrete units whose borders coincide with habitat breakages. This complex structure seems to be shaped by the geo-morphological and ecological features of the landscape, through a mechanism of local specialization of the resident dolphins. Five distinct clusters were identified in the (meta)population and two of them were solid enough to be further investigated and compared. Significant differences were found in the network parameters, suggesting a different social organization of the clusters, possibly as a consequence of the different local specialization.     

Gestational diabetes mellitus (GDM) decreases butyrylcholinesterase (BChE) activity and changes its relationship with lipids

Many conditions interfere with butyrylcholinesterase (BChE) activity, e.g., pregnancy or presence of the BCHE gene variant −116A can decrease activity whereas obesity and types I and II diabetes mellitus can increase activity. In this study, we examined BChE activity, −116A and 1615A BCHE gene variants, and anthropometric and biochemical variables associated with diabetes in patients with gestational diabetes mellitus (GDM) and in healthy pregnant women. BChE activity was measured spectrophotometrically using propionylthiocholine as substrate and genotyping of the −116 and 1615 sites of the BCHE gene was done with a TaqMan SNP genotyping assay. Three groups were studied: 150 patients with GDM, 295 healthy pregnant women and 156 non-pregnant healthy women. Mean BChE activity was significantly lower in healthy pregnant women than in women from the general population and was further reduced in GDM patients. BChE activity was significantly reduced in carriers of −116A in GDM patients and healthy pregnant women. Although GDM patients had a significantly higher mean body mass index (BMI) and triglycerides than healthy pregnant women, they had lower mean BChE activity, suggesting that the lowering effect of GDM on BChE activity was stronger than the characteristic enhancing effect of increased BMI and triglycerides.     

Fumarate and cytosolic pH as modulators of the synthesis or consumption of C4 organic acids through NADP-malic enzyme in Arabidopsis thaliana

Arabidopsis thaliana is a plant species that accumulates high levels of organic acids and uses them as carbon, energy and reducing power sources. Among the enzymes that metabolize these compounds, one of the most important ones is malic enzyme (ME). A. thaliana contains four malic enzymes (NADP-ME 1–4) to catalyze the reversible oxidative decarboxylation of malate in the presence of NADP. NADP-ME2 is the only one located in the cell cytosol of all Arabidopsis organs providing most of the total NADP-ME activity. In the present work, the regulation of this key enzyme by fumarate was investigated by kinetic assays, structural analysis and a site-directed mutagenesis approach. The final effect of this metabolite on NADP-ME2 forward activity not only depends on fumarate and substrate concentrations but also on the pH of the reaction medium. Fumarate produced an increase in NADP-ME2 activity by binding to an allosteric site. However at higher concentrations, fumarate caused a competitive inhibition, excluding the substrate malate from binding to the active site. The characterization of ME2-R115A mutant, which is not activated by fumarate, confirms this hypothesis. In addition, the reverse reaction (reductive carboxylation of pyruvate) is also modulated by fumarate, but in a different way. The results indicate pH-dependence of the fumarate modulation with opposite behavior on the two activities analyzed. Thereby, the coordinated action of fumarate over the direct and reverse reactions would allow a precise and specific modulation of the metabolic flux through this enzyme, leading to the synthesis or degradation of C₄ compounds under certain conditions. Thus, the physiological context might be exerting an accurate control of ME activity in planta, through changes in metabolite and substrate concentrations and cytosolic pH.