A demonstration of an affinity between pyrite and organic matter in a hydrothermal setting

One of the key-principles of the iron-sulphur world theory is to bring organic molecules close enough to interact with each other, using the surface of pyrite as a substrate in a hydrothermal setting. The present paper explores the relationship of pyrite and organic matter in a hydrothermal setting from the geological record; in hydrothermal calcite veins from Carboniferous limestones in central Ireland. Here, the organic matter is accumulated as coatings around, and through, pyrite grains. Most of the pyrite grains are euhedral-subhedral crystals, ranging in size from ca 0.1-0.5 mm in diameter, and they are scattered throughout the matrix of the vein calcite. The organic matter was deposited from a hydrothermal fluid at a temperature of at least 200°C, and gives a Raman signature of disordered carbon. This study points to an example from a hydrothermal setting in the geological record, demonstrating that pyrite can have a high potential for the concentration and accumulation of organic materials

Characterization and genetic diversity in Paspalum notatum Flügge accessions: Morphological and geographical distance

The objective of this work was to describe morphology and grouping of Paspalum notatum accessions, based on multicategorical data which discards the redundant variables for quantification of genetic diversity. We also tested the hypothesis that geographical distance was correlated with morphological divergence. In our study, multivariate analyzes successfully demonstrated the geographic and morphological variability of the P. notatum accessions characterized. Many of these evaluated accessions can be included in future genetic improvement programs. Based on two methodologies for discarding variables, it was possible to identify the potentially important morphological characteristics from genetic diversity studies and characterize new accessions aimed at improving forage and seed production. The methodologies used to discard variables are biometric tools that can be used successfully in future plant breeding programs, especially when a large number of traits and accessions are being evaluated. Although significant, geographic distance had a low association with morphological traits. This indicated the need to use other characteristics, such as forage and seed yield, in addition to molecular analysis. Our analyzes showed genetic variability in P. notatum for all the characteristics studied

Oroidin Inhibits the Activity of the Multidrug Resistance Target Pdr5p from Yeast Plasma Membranes

Oroidin was isolated from the marine sponge Agelas sventres and inhibited the activity and function of Pdr5p, an enzyme responsible for the multidrug resistance phenotype in Saccharomyces cerevisiae. This compound may help in the development of new drugs that reverse this dangerous phenotype of pathogenic yeast and fungi.CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior)Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ)FAPERJ (Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro)CENPES-PetrobrasCENPES-PetrobrasFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP[05/60175-2

21-Benzylidene Digoxin: A Proapoptotic Cardenolide of Cancer Cells That Up-Regulates Na,K-ATPase and Epithelial Tight Junctions

<div><p>Cardiotonic steroids are used to treat heart failure and arrhythmia and have promising anticancer effects. The prototypic cardiotonic steroid ouabain may also be a hormone that modulates epithelial cell adhesion. Cardiotonic steroids consist of a steroid nucleus and a lactone ring, and their biological effects depend on the binding to their receptor, Na,K-ATPase, through which, they inhibit Na⁺ and K⁺ ion transport and activate of several intracellular signaling pathways. In this study, we added a styrene group to the lactone ring of the cardiotonic steroid digoxin, to obtain 21-benzylidene digoxin (21-BD), and investigated the effects of this synthetic cardiotonic steroid in different cell models. Molecular modeling indicates that 21-BD binds to its target Na,K-ATPase with low affinity, adopting a different pharmacophoric conformation when bound to its receptor than digoxin. Accordingly, 21-DB, at relatively high µM amounts inhibits the activity of Na,K-ATPase α₁, but not α₂ and α₃ isoforms. In addition, 21-BD targets other proteins outside the Na,K-ATPase, inhibiting the multidrug exporter Pdr5p. When used on whole cells at low µM concentrations, 21-BD produces several effects, including: 1) up-regulation of Na,K-ATPase expression and activity in HeLa and RKO cancer cells, which is not found for digoxin, 2) cell specific changes in cell viability, reducing it in HeLa and RKO cancer cells, but increasing it in normal epithelial MDCK cells, which is different from the response to digoxin, and 3) changes in cell-cell interaction, altering the molecular composition of tight junctions and elevating transepithelial electrical resistance of MDCK monolayers, an effect previously found for ouabain. These results indicate that modification of the lactone ring of digoxin provides new properties to the compound, and shows that the structural change introduced could be used for the design of cardiotonic steroid with novel functions.</p></div

21-BD regulates tight junctions.

<p>MDCK cells were cultured in transwell permeable supports and treated with 5, 10 and 50 µM 21-BD. (A) TER was measured as a function of time. The control TER data (white circles, dotted line) averaged 183±8 Ω.cm² (n = 13) and were normalized to 100%. 5 and 10 µM 21-BD provoke transient small increases of TER, while 50 µM 21-BD causes a stronger and a sustained TER increase (red circles). (B) MDCK cells were incubated 48 h with different concentrations of 21-BD (red symbols) or digoxin (green symbols). mRNA cell content of claudins -4 (circles) and -2 (triangles) were measured by quantitative real time PCR. (C) Protein cell content of the tight junction integral membrane proteins claudins -4 and -2 and the membrane-associated protein ZO-1 as a function of 21-BD concentration in the media for 48 h. Images from the left part of the figure C are representative immunoblots and the graph in the right part is the statistical analysis.</p

21-BD induces apoptosis in HeLa and CHO-K1 cells.

<p>(A) Score value obtained from the comet assay of CHO-K1 cells incubated 24 h with 21-BD at different concentrations (red circles). (B) Micronucleated cells percentage of CHO-K1 cultures incubated with 21-BD at different concentrations for 24 h. A 24 h incubation with 0.4 mM Methyl methanesulfonate (MMS) was used as a control (A, B, blue circles). (C) Apoptotic and necrotic HeLa cells after 24 h of incubation in control media (white bars), media with 50 µM 21-BD (red bars) or 2 µM digoxin (green bars) for 24 h. Apoptosis and necrosis were detected by flow cytometry ussing an annexin-V translocation assay and the incorporation of propidium iodide in to the nucleus, respectively. <i>P</i><0.01.</p

21-BD effect on Na,K-ATPase and Pdr5p activity.

<p>(A) 21-BD competition of ³H-ouabain binding on HeLa cells; the control for maximal binding is represented with a white circle and a long dashed line, competition of ouabain and 21-BD is shown with blue and red circles respectively. (B) Inhibition of rat´s brain hemisphere Na,K-ATPase after 2 h incubation with digoxin (green circles) or 21-BD (red circles). (C) Effect of 21-BD on the Na,K-ATPase activity on proteins expressed in Sf9 insect cells, Na,K-ATPase activity was measured on Sf9 cells expressing the rat α₁ β₁ (orange circles) or β₁ (red circles) after 15 min treatment with the indicated concentrations of 21-BD. (D) Dose-response curve for the effects of 21-DB on Na,K-ATPase activity of mouse kidney membrane preparations. E) Effect of 21-BD (red circles) or digoxin (green circles) on the activity of the Pdr5p transporter.</p

21-BD increases Na,K-ATPase expression in cancer cells.

<p>(A) Na,K-ATPase activity after incubation of HeLa cells with 21-BD or digoxin for 48 h with different concentrations of 21-BD. B) mRNA content of the Na,K-ATPase α₁ and β₁ subunits of HeLa cells after 48 h incubation with various concentrations of 21-BD.</p

High concentrations of 21-BD reduce cell viability of HeLa and RKO.

<p>HeLa (A) or RKO (B) cells were treated with digoxin (green symbols) or 21-BD (red symbols) for 24 (circles) or 48 (squares) h. Viability was measured by MTT reduction assay. 100 and 25 µM 21-BD induced the statistically significant reduction of HeLa and RKO viability, respectively (p<0.0084). Digoxin reduces HeLa viability starting with 150 µM for 24 h and 50 µM for 48 h (p<0.001). RKO cells have a higher sensitivity to digoxin that induces statistically significant differences starting from 1.6 µM for 48 h (p<0.0001).</p