João Coimbra

Polycyclic aromatic hydrocarbons inhibit in vitro ovarian steroidogenesis in the flounder (Platichthys flesus L.)

The in vitro effect of polycyclic aromatic hydrocarbons (PAHs) on ovarian steroidogenesis of the flounder (Platichthys flesus L.) was determined. Fully vitellogenic ovary tissue was in vitro incubated in the presence of phenanthrene, benzo[a]pyrene or chrysene, using 17alpha-hydroxyprogesterone or androstenedione as precursors. Androstenedione (A), testosterone (T) and 17beta-estradiol (E(2)) synthesised in the presence of PAHs were assayed by radioimmunoassay and results compared with control incubations. In order to establish the effect of PAHs on the steroidogenic enzyme systems cytochrome P450 17,20-lyase (P450-17,20l), 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and cytochrome P450 aromatase (P450-arom), results were also compared with the action of ketoconazole (KCZ) and aminoglutethimide (AMG), wich are, respectively, inhibitors of cytochrome P450 steroidogenic enzymes and of P450-arom. KCZ inhibited secretion of A and E(2) in 65% and T in 40%, as a consequence of inhibited P450-17,20l and P450-arom. AMG inhibited P450-arom, which resulted in decreased E(2) synthesis to approximately 50% of control incubations. All the three PAHs inhibited A secretion by approximately 50% and E(2) from 10 to 40%. Because steroid conjugation was also inhibited by phenanthrene, it could be concluded that PAH action was mediated by an inhibitory effect over P450-17,20l, 17beta-HSD and P450-arom. Except for 17beta-HSD, PAHs resembled KCZ, and P450-17,20l was the most sensitive to their inhibitory effect. In conclusion, PAHs strongly blocked the activity of P450-17,20l, a rate-limiting enzyme for conversion of C21 to C19 steroids, and showed, therefore, the potential to disrupt the reproductive cycle of fish living in polluted environments, due to impairment of steroid biosynthesis.

Protein-Ligand Docking in the New Millennium – A Retrospective of 10 Years in the Field

Protein-ligand docking is currently an important tool in drug discovery efforts and an active area of research that has been the subject of important developments over the last decade. These are well portrayed in the rising number of available protein-ligand docking software programs, increasing level of sophistication of its most recent applications, and growing number of users. While starting by summarizing the key concepts in protein-ligand docking, this article presents an analysis of the evolution of this important field of research over the past decade. Particular attention is given to the massive range of alternatives, in terms of protein-ligand docking software programs currently available. The emerging trends in this field are the subject of special attention, while old established docking alternatives are critically revisited. Current challenges in the field of protein-ligand docking such as the treatment of protein flexibility, the presence of structural water molecules and its effect in docking, and the entropy of binding are dissected and discussed, trying to anticipate the next years in the field.

Is there a compromise between nutrient uptake and gas exchange in the gut of Misgurnus anguillicaudatus, an intestinal air-breathing fish?

The Asian weatherloach, Misgurnus anguillicaudatus (Cobitidae), is a facultative air-breathing teleost fish that makes use of its hindgut or intestine as an accessory air-breathing organ (ABO). The hindgut is highly modified, being well vascularized with intraepithelial capillaries, which makes it well suited for gas exchange. However, the consequences for nutrient uptake, the traditional function of the intestine are unknown. The alimentary canal was examined histologically to assess differences between the fore-, mid- and hindgut regions that have been considered as the digestive, spiral and respiratory zones, respectively. In order to characterise the potential digestive (absorptive) function of the respiratory zone we used semi-quantitative polymerase chain reaction (PCR) to detect the presence of the intestinal Na(+):glucose cotransporter (SGLT1; SLC5A1) and H(+):peptide cotransporter (PEPT1a; SLC15A1) and partially sequenced the SGLT1 and PEPT1a cDNAs. These two transporters play important roles in the absorption of carbohydrate and di-/tripeptides, respectively, in the gut of fishes and other vertebrates and were therefore used as markers for potential nutrient uptake function. We also determined their tissue distributions through semi-quantitative RT-PCR. The effects of diet composition (high protein or high carbohydrate) or fasting on gene expression were also examined. SGLT1 expression was found in kidney, liver, heart, as well as in the three zones of the gut except the most distal part of the hindgut. PEPT1a mRNA was found in heart, brain, liver, and fore- and midgut, but absent in the hindgut. Our results clearly show high expression of SGLT1 (both mRNA and protein by immunolocalization) and PEPT1a (mRNA) in the foregut and midgut correlated with the digestive region of the gut. Modulatory effects of diet on the gene expression for both SGLT1 and PEPT1a were not observed. The presence of SGLT1 transcripts in the respiratory zone of the intestine suggests an overlap in function. However, in the case of PEPT1a, the distal limit was the midgut. Thus, despite its highly modified structure, the hindgut of the loach retains some potential nutrient uptake function.

Comparative analysis of the performance of commonly available density functionals in the determination of geometrical parameters for zinc complexes.

In this study, a set of 50 transition‐metal complexes of Cu(I) and Cu(II), were used in the evaluation of 18 density functionals in geometry determination. In addition, 14 different basis sets were considered, including four commonly used Poples all‐electron basis sets; four basis sets including popular types of effective‐core potentials: Los Alamos, Steven‐Basch‐Krauss, and Stuttgart‐Dresden; and six triple‐ζ basis sets. The results illustrate the performance of different methodological alternatives for the treatment of geometrical properties in relevant copper complexes, pointing out Double‐Hybrid (DH) and Long‐range Correction (LC) Generalized Gradient Approximation (GGA) methods as better descriptors of the geometry of the evaluated systems. These however, are associated with a computational cost several times higher than some of the other methods employed, such as the M06 functional, which has also demonstrated a comparable performance. Regarding the basis sets, 6–31+G(d) and 6–31+G(d,p) were the best performing approaches. In addition, the results show that the use of effective‐core potentials has a limited impact, in terms of the accuracy in the determination of metal‐ligand bond‐lengths and angles in our dataset of copper complexes. Hence, these could become a good alternative for the geometrical description of these systems, particularly CEP‐121G and SDD basis sets, if one is considering larger copper complexes where the computational cost could be an issue.

P2X7-induced zeiosis promotes osteogenic differentiation and mineralization of postmenopausal bone marrow-derived mesenchymal stem cells

Polymorphisms of the P2X7 receptor have been associated with increased risk of fractures in postmenopausal women. Although both osteoblasts and osteoclasts express P2X7 receptors, their function in osteogenesis remains controversial. Here, we investigated the role of the P2X7 receptor on osteogenic differentiation and mineralization of bone marrow mesenchymal stem cell (BMSC) cultures from postmenopausal women (age 71 ± 3 yr, n=18). We focused on the mechanisms related to intracellular [Ca2+]i oscillations and plasma membrane‐dynamics. ATP, and the P2X7 agonist BzATP (100 μM), increased [Ca2+]i in parallel to the formation of membrane pores permeable to TO‐PRO‐3 dye uptake. ATP and BzATP elicited reversible membrane blebs (zeiosis) in 38 ± 1 and 70 ± 1% of the cells, respectively. P2X7‐induced zeiosis was Ca2+ independent, but involved phospholipase C, protein kinase C, and Rho‐kinase activation. BzATP (100 μM) progressively increased the expression of Runx‐2 and Osterix transcription factors by 452 and 226% (at d 21), respectively, alkaline phosphatase activity by 88% (at d 28), and mineralization by 329% (at d 43) of BMSC cultures in a Rho‐kinase‐dependent manner. In summary, reversible plasma membrane zeiosis involving cytoskeleton rearrangements due to activation of the P2X7‐Rho‐kinase axis promotes osteogenic differentiation and mineralization of BMSCs, thus providing new therapeutic targets for postmenopausal bone loss.—Noronha‐Matos, J. B., Coimbra, J., Sá‐e‐Sousa, A., Rocha, R., Marinhas, J., Freitas, R., Guerra‐Gomes, S., Ferreirinha, F., Costa, M. A., Correia‐de‐Sá, P., P2X7‐induced zeiosis promotes osteogenic differentiation and mineralization of postmenopausal bone marrow‐derived mesenchymal stem cells FASEB J. 28, 5208–5222 (2014). www.fasebj.org

Branchial ammonia excretion in the Asian weatherloach Misgurnus anguillicaudatus

The weatherloach, Misgurnus anguillicaudatus, is a freshwater, facultative air-breathing fish that lives in streams and rice paddy fields, where it may experience drought and/or high environmental ammonia (HEA) conditions. The aim of this study was to determine what roles branchial Na(+)/K(+)-ATPase, H(+)-ATPase, and Rhcg have in ammonia tolerance and how the weatherloach copes with ammonia loading conditions. The loachs high ammonia tolerance was confirmed as was evident from its high 96 h LC(50) value and high tissue tolerance to ammonia. The weatherloach does not appear to make use of Na(+)/NH(4)(+)-ATPase facilitated transport to excrete ammonia when exposed to HEA or to high environmental pH since no changes in activity were observed. Using immunofluorescence microscopy, distinct populations of vacuolar (V)-type H(+)-ATPase and Na(+)/K(+)-ATPase immunoreactive cells were identified in branchial epithelia, with apical and basolateral staining patterns, respectively. Rhesus C glycoprotein (Rhcg1), an ammonia transport protein, immunoreactivity was also found in a similar pattern as H(+)-ATPase. Rhcg1 (Slc42a3) mRNA expression also increased significantly during aerial exposure, although not significantly under ammonia loading conditions. The colocalization of H(+)-ATPase and Rhcg1 to the similar non-Na(+)/K(+)-ATPase immunoreactive cell type would support a role for H(+)-ATPase in ammonia excretion via Rhcg by NH(4)(+) trapping. The importance of gill boundary layer acidification in net ammonia excretion was confirmed in this fish; however, it was not associated with an increase in H(+)-ATPase expression, since tissue activity and protein levels did not increase with high environmental pH and/or HEA. However the V-ATPase inhibitor, bafilomycin, did decrease net ammonia flux whereas other ion transport inhibitors (amiloride, SITS) had no effect. H(+)-ATPase inhibition also resulted in a consequent elevation in plasma ammonia levels and a decrease in the net acid flux. In gill, aerial exposure was also associated with a significant increase in membrane fluidity (or increase in permeability) which would presumably enhance NH(3) permeation through the plasma membrane. Taken together, these results indicate the gill of the weatherloach is responsive to aerial conditions that would aid ammonia excretion.

Reproductive cycle, ovarian development, and vertebrate-type steroids profile in the freshwater prawn Macrobrachium rosenbergii

Abstract This study is intended to characterize the ovarian development and hemolymph vertebrate-type steroids concentration during the reproductive cycle of the freshwater prawn, Macrobrachium rosenbergii. A five-stage classification based on the external observation of the ovarys size and color as seen through the tegument was used. The results showed the existence of a direct correspondence between the ovarian stages and the gonadosomatic index, the oöcyte diameter, and the characteristics of ovarian histology. In each stage total bleeding of the prawns was conducted and the hemolymph concentrations of 17β-estradiol (E2), testosterone (T) and 17α-hydroxyprogesterone (17-OHP; conjugated and unconjugated) were determined by solid-phase radioimmunoassay (RIA). High levels of unconjugated 17-OHP, relatively constant concentrations of unconjugated T, and null concentration of unconjugated E2 were found throughout the five stages considered. Nonetheless, low levels of E2 were determined in all stages, in conjugated (glucoronide) form. The highest levels were obtained in stage II and III (18.14 ± 14.52 pg/ml hemolymph) and progressively declined in the other stages (7.53 ± 6.76 pg/ml hemolymph). These results point out the possible involvement of vertebrate-type steroids in the endocrine regulation of the Macrobrachium rosenbergiis ovarian cycle. To our knowledge this is the first study concerning the vertebrate-like steroid levels throughout the ovarian cycle and is a step needed to characterize the hemolymph profile of these steroids in order to elucidate the possible role in the regulation of the reproductive crustacean cycle.

Haemolymph Unconjugated and Conjugated Steroids During Reproduction in Penaeus Japonicus (Crustacea: Decapoda)

With reversed-phase high performance liquid chromatography (HPLC) with diode array detection, the steroid and ecdysteroid contents have been studied of haemolymph of Penaeus japonicus during the reproductive cycle. In females, conjugated androgens and conjugated and unconjugated estrogens arc present, some occurring mainly in vitellogenic stages, others in immature or post-spawning animals. In males with quiescent testes, during the winter months only unconjugated estrogens occur. Also unconjugated cortisol is present in this period, in both males and females. The presence of a large amount of unknown steroids suggests the existence in crustaceans of metabolic pathways different from those in vertebrates.

Relevant interactions of antimicrobial iron chelators and membrane models revealed by nuclear magnetic resonance and molecular dynamics simulations.

The dynamics and interaction of 3-hydroxy-4-pyridinone fluorescent iron chelators, exhibiting antimicrobial properties, with biological membranes were evaluated through NMR and molecular dynamics simulations. Both NMR and MD simulation results support a strong interaction of the chelators with the lipid bilayers that seems to be strengthened for the rhodamine containing compounds, in particular for compounds that include ethyl groups and a thiourea link. For the latter type of compounds the interaction reaches the hydrophobic core of the lipid bilayer. The molecular docking and MD simulations performed for the potential interaction of the chelators with DC-SIGN receptors provide valuable information regarding the cellular uptake of these compounds since the results show that the fluorophore fragment of the molecular framework is essential for an efficient binding. Putting together our previous and present results, we put forward the hypothesis that all the studied fluorescent chelators have access to the cell, their uptake occurs through different pathways and their permeation properties correlate with a better access to the cell and its compartments and, consequently, with the chelators antimicrobial properties.

New Parameters for Higher Accuracy in the Computation of Binding Free Energy Differences upon Alanine Scanning Mutagenesis on Protein–Protein Interfaces

Knowing how proteins make stable complexes enables the development of inhibitors to preclude protein-protein (P:P) binding. The identification of the specific interfacial residues that mostly contribute to protein binding, denominated as hot spots, is thus critical. Here, we refine an in silico alanine scanning mutagenesis protocol, based on a residue-dependent dielectric constant version of the Molecular Mechanics/Poisson-Boltzmann Surface Area method. We have used a large data set of structurally diverse P:P complexes to redefine the residue-dependent dielectric constants used in the determination of binding free energies. The accuracy of the method was validated through comparison with experimental data, considering the per-residue P:P binding free energy (ΔΔGbinding) differences upon alanine mutation. Different protocols were tested, i.e., a geometry optimization protocol and three molecular dynamics (MD) protocols: (1) one using explicit water molecules, (2) another with an implicit solvation model, and (3) a third where we have carried out an accelerated MD with explicit water molecules. Using a set of protein dielectric constants (within the range from 1 to 20) we showed that the dielectric constants of 7 for nonpolar and polar residues and 11 for charged residues (and histidine) provide optimal ΔΔGbinding predictions. An overall mean unsigned error (MUE) of 1.4 kcal mol-1 relative to the experiment was achieved in 210 mutations only with geometry optimization, which was further reduced with MD simulations (MUE of 1.1 kcal mol-1 for the MD employing explicit solvent). This recalibrated method allows for a better computational identification of hot spots, avoiding expensive and time-consuming experiments or thermodynamic integration/ free energy perturbation/ uBAR calculations, and will hopefully help new drug discovery campaigns in their quest of searching spots of interest for binding small drug-like molecules at P:P interfaces.