Metabolites from the Euryhaline Ciliate Pseudokeronopsis erythrina

TThree new secondary metabolites (named erythrolactones A2, B2 and C2), that are characterized by a central 4‐hydroxy‐unsaturated δ lactone ring bearing an alkyl saturated chain at C(2) and a butyl‐benzenoid group at C(5), together with their respective sulfate esters (erythrolactones A1, B1 and C1), have been isolated from cell cultures of Pseudokeronopsis erythrina, clone TL‐1. The structures are assigned on the basis of extensive spectroscopic measurements (1D and 2D NMR, UV, IR and HR‐MALDI‐TOF). A plausible biogenetic route for their formation is also suggested. Cold‐shock treatment was performed in order to induce the discharge of the metabolites contained in pigment granules lying on the ciliary organelles of this microorganism. HPLC‐ESI‐MS analysis of this granule discharge reveals that erythrolactones A2–C2 are actually therein contained, strongly suggesting a possible role for these metabolites in the chemical defence strategy of P. erythrina

Improving the phloroglucinolysis protocol and characterization of Sagrantino wines proanthocyanidins

4openInternationalProanthocyanidins are key metabolites that explain wine sensorial character (bitterness and astringency) and red wine color changes during aging. Therefore, a fast and accurate method to evaluate the degree of polymerization and the structural composition of the polymeric proanthocyanidins is a crucial analytical tool. Phloroglucinolysis is the most used method for this analysis but, unfortunately, the phloroglucinol adducts of the monomeric flavan-3-ols are not commercially available, making the results less accurate. The aim of this work was the isolation by semi-preparative high performance liquid chromatography (HPLC) of these non-commercial compounds and their use for the development of an accurate UHPLC-MS/MS protocol. The purity of each adduct was established via quantitative 1H-nuclear magnetic resonance (NMR) measurements with 3-trimethylsilyl-propionic-d4 acid sodium salt as the calibration standard. The developed method was applied to evaluate the proanthocyanidins profile of Sagrantino di Montefalco wines in comparison to other well-known tannic wines. Commercial, 6–8 years old Sagrantino wines were demonstrated to be very rich in epicatechin type B procyanidins, to have low galloylation %, and to have a high mean degree of polymerization of the proanthocyanidins with respect to the other analyzed winesopenArapitsas, P.; Perenzoni, D.; Guella, G.; Mattivi, F.Arapitsas, P.; Perenzoni, D.; Guella, G.; Mattivi, F

Natural Products among Brown Algae: The Case of Cystoseira schiffneri HAMEL (Sargassaceae, Phaeophyceae)

A chemotaxonomic study on the marine brown alga Cystoseira schiffneri collected from the Tunisian marine coast allowed us to identify kjellmanianone (1) and a new isololiolide derivative named schiffnerilolide (2). The structu re elucidati on and the assignment of relative con figurations of the isolated natur al products were based on advanced mass spectrometric and nuclear magnetic resonance techniques. This outcome suggested a close phylogenetic relationship of C. schiffneri with brown algae bel onging to genus Sargassum C. A GARDH. Molecular characterization using the nuclear small subunit rRNA (SSU rRNA) gene (18S) sequence as genetic marker was made. Pigme nt analysis showed a significant seasonal change of carotenoids, in particular of fucoxanthin and fucoxanthinol. Also galactolipids, the main constituents of the thylakoid membranes, showed remarkable seasonal changes

H/D exchange processes in flavonoids: kinetics and mechanistic investigations

Several classes of flavonoids, such as anthocyanins, flavonols, flavanols, and flavones, undergo a slow H/D exchange on aromatic ring A, leading to full deuteration at positions C(6) and C(8). Within the flavanol class, H-C(6) and H-C(8) of catechin and epicatechin are slowly exchanged in D2O to the corresponding deuterated analogues. Even quercetin, a relevant flavonol representative, shows the same behaviour in a D2O/DMSOd6 1:1 solution. Detailed kinetic measurements of these H/D exchange processes are here reported by exploiting the time-dependent changes of their peak areas in the 1H-NMR spectra taken at different temperatures. A unifying reaction mechanism is also proposed based on our detailed kinetic observations, even taking into account pH and solvent effects. Molecular modelling and QM calculations were also carried out to shed more light on several molecular details of the proposed mechanis

Cytotoxic effects and apoptotic signalling mechanisms of the sesquiterpenoid euplotin C, a secondary metabolite of the marine ciliate Euplotes crassus, in tumour cells

Most antitumour agents with cytotoxic properties induce apoptosis. The lipophilic compound euplotin C, isolated from the ciliate Euplotes crassus, is toxic to a number of different opportunistic or pathogenic microorganisms, although its mechanism of action is currently unknown. We report here that euplotin C is a powerful cytotoxic and pro-apoptotic agent in mouse AtT-20 and rat PC12 tumour-derived cell lines. In addition, we provide evidence that euplotin C treatment results in rapid activation of ryanodine receptors, depletion of Ca2+ stores in the endoplasmic reticulum (ER), the release of cytochrome c from the mitochondria, activation of caspase-12, and activation of caspase-3, leading to apoptosis. Intracellular Ca2+ overload is an early event which induces apoptosis and is parallelled by ER stress and the release of cytochrome c, whereas caspase-12 may be activated by euplotin C at a later stage in the apoptosis pathway. These events, either independently or concomitantly, lead to the activation of the caspase-3 and its downstream effectors, triggering the cell to undergo apoptosis. These results demonstrate that euplotin C may be considered for the design of cytotoxic and pro-apoptotic new drugs.L'articolo è disponibile sul sito dell'editore http://www.springerlink.com

Molecular mechanisms of euplotin C-induced apoptosis: involvement of mitochondrial dysfunction, oxidative stress and proteases

The metabolite euplotin C (EC), isolated from the marine ciliate Euplotes crassus, is a powerful cytotoxic and pro-apoptotic agent in tumour cell lines. For instance, EC induces the rapid depletion of ryanodine Ca2+ stores, the release of cytochrome c from the mitochondria, and the activation of caspase-3, leading to apoptosis. The purpose of this study was to gain further insight into the mechanisms of EC-induced apoptosis in rat pheochromocytoma PC12 cells. We found that EC increases Bax/Bcl-2 ratio and that Bax is responsible of the EC-induced dissipation of the mitochondrial membrane potential (Δψm). In addition, EC induces the generation of reactive oxygene species (ROS) without involvement of p53. The inhibition of ROS generation prevents, at least in part, the pro-apoptotic effects of EC as well as the effects of EC on Bax, Δψm and intracellular free Ca2+, indicating a cross-talk between different pathways. However, definition of the effector cascade turns out to be more complex than expected and caspase-independent mechanisms, acting in parallel with caspases, should also be considered. Among them, EC increases the expression/activity of calpains downstream of ROS generation, although calpains seem to exert protective effects.L'articolo è disponibile sul sito dell'editore http://www.springerlink.co

Structures, Biological Activities and Phylogenetic Relationships of Terpenoids from Marine Ciliates of the Genus Euplotes

In the last two decades, large scale axenic cell cultures of the marine species comprising the family Euplotidae have resulted in the isolation of several new classes of terpenoids with unprecedented carbon skeletons including the (i) euplotins, highly strained acetylated sesquiterpene hemiacetals; (ii) raikovenals, built on the bicyclo[3.2.0]heptane ring system; (iii) rarisetenolides and focardins containing an octahydroazulene moiety; and (iv) vannusals, with a unique C30 backbone. Their complex structures have been elucidated through a combination of nuclear magnetic resonance spectroscopy, mass spectrometry, molecular mechanics and quantum chemical calculations. Despite the limited number of biosynthetic experiments having been performed, the large diversity of ciliate terpenoids has facilitated the proposal of biosynthetic pathways whereby they are produced from classical linear precursors. Herein, the similarities and differences emerging from the comparison of the classical chemotaxonomy approach based on secondary metabolites, with species phylogenesis based on genetic descriptors (SSU-rDNA), will be discussed. Results on the interesting ecological and biological properties of ciliate terpenoids are also reported

The non-proteic extrusive secondary metabolites in ciliated protists

The non-proteic extrusive secondary metabolites in ciliated protists F. Buonanno1, A. Anesi2, G. Guella2, E. Marcantoni3, S. Giorgi3, C. Ortenzi1 1Laboratory of Protistology and Biology Education, University of Macerata, 62100 Macerata, Italy 2Bioorganic Chemistry Laboratory, Department of Physics, University of Trento, 38050 Povo, Trento, Italy 3School of Sciences and Technologies, Section of Chemistry, University of Camerino, 62032 Camerino, Macerata, Italy Extrusomes are membrane-bound ejectable organelles widely distributed in protists. They are usually localized in the cell cortex and attached to the cell membrane, and they are able to discharge their contents to the outside of the cell in response to mechanical or chemical stimuli. Notably, cells that discharge their extrusomes remains intact and functional. The chemical nature of protists\u2019 extrusive compounds characterized to date is extremely variable, including proteins, carbohydrates, lipids, and dozens of additional classes of secondary metabolites. However an increasing set of data are now available for particular group of protists, the ciliated protozoa. Many of non-proteic extrusive secondary metabolites in ciliates function for chemical offense or defense in prey-predator interactions against unicellular or/and multicellular organisms. It is worthy of note that at least some of these secondary metabolites have been demonstrated to show antibiotic, anti-cancer and pro-apoptotic properties in addition to their physiological functions. Among these compounds, euplotin C produced by the ciliate Euplotes crassus, and climacostol produced by Climacostomum virens, have been shown to activate programmed cell death by impairing mitochondrial membrane potential and inducing ROS generation in mammalian tumor cell lines. Interestingly, an antimicrobial activity against Gram-positive bacteria and fungal pathogens was also demonstrated for climacostol. Overall, in addition to the understanding of their physiological and ecological functions, the study of non-proteic secondary metabolites of ciliated protozoa may set the basis for the development of a novel series of antitumor and antimicrobial agents

Vibrational properties of inclusion complexes: the case of indomethacin-cyclodextrin

Vibrational properties of inclusion complexes with cyclodextrins are studied by means of Raman spectroscopy and numerical simulation. In particular, Raman spectra of the non-steroidal, anti-inflammatory drug indomethacin undergo notable changes in the energy range between 1600 and 1700 cm$^{-1}$ when inclusion complexes with cyclodextrins are formed. By using both \emph{ab initio} quantum chemical calculations and molecular dynamics, we studied how to relate such changes to the geometry of the inclusion process, disentangling single-molecule effects, from changes in the solid state structure or dimerization processes.Comment: 14 file figure

Pore Formation by Equinatoxin II, a Eukaryotic Protein Toxin, Occurs by Induction of Nonlamellar Lipid Structures

Pore formation in the target cell membranes is a common mechanism used by many toxins in order to kill cells. Among various described mechanisms, a toroidal pore concept was described recently in the course of action of small antimicrobial peptides. Here we provide evidence that such mechanism may be used also by larger toxins. Membrane-destabilizing effects of equinatoxin II, a sea anemone cytolysin, were studied by various biophysical techniques. 31P NMR showed an occurrence of an isotropic component when toxin was added to multilamellar vesicles and heated. This component was not observed with melittin, alpha-staphylococcal toxin, or myoglobin. It does not originate from isolated small lipid structures, since the size of the vesicles after the experiment was similar to the control without toxin. Electron microscopy shows occurrence of a honeycomb structure, previously observed only for some particular lipid mixtures. The analysis of FTIR spectra of the equinatoxin II-lipid complex showed lipid disordering that is consistent with isotropic component observed in NMR. Finally, the cation selectivity of the toxin-induced pores increased in the presence of negatively charged phosphatidic acid, indicating the presence of lipids in the conductive channel. The results are compatible with the toroidal pore concept that might be a general mechanism of pore formation for various membrane-interacting proteins or peptides