Conformation of 1,4-dihydropyridine — planar or boat-like?

AbstractThe geometry of the 1,4-dihydropyridine molecule was completely optimized employing three different ab initio basis sets (6–31 G*, 4–31 G, STO—3G). The most reliable 6–31G* basis set provides a very flat boat conformation which may easily undergo defolding to a planar ring arrangement. This result is discussed with respect to enzymatic redox cofactors and the pharmacological activity of dihydropyridine calcium antagonists

Potent Antioxidant and Genoprotective Effects of Boeravinone G, a Rotenoid Isolated from Boerhaavia diffusa

Background and Aims: Free radicals are implicated in the aetiology of some gastrointestinal disorders such as gastric ulcer, colorectal cancer and inflammatory bowel disease. In the present study we investigated the antioxidant and genoprotective activity of some rotenoids (i.e. boeravinones) isolated from the roots of Boerhaavia diffusa, a plant used in the Ayurvedic medicine for the treatment of diseases affecting the gastrointestinal tract. Methods/Principal Findings: Antioxidant activity has been evaluated using both chemical (Electron Spin Resonance spectroscopy, ESR) and Caco-2 cells-based (TBARS and ROS) assays. DNA damage was evaluated by Comet assay, while pERK 1/2 and phospho-NF-kB p65 levels were estimated by western blot. Boeravinones G, D and H significantly reduced the signal intensity of ESR induced by hydroxyl radicals, suggesting a scavenging activity. Among rotenoids tested, boeravinone G exerted the most potent effect. Boeravinone G inhibited both TBARS and ROS formation induced by Fenton's reagent, increased SOD activity and reduced H 2O 2-induced DNA damage. Finally, boeravinone G reduced the levels of pERK 1 and phospho-NF-kB p65 (but not of pERK 2) increased by Fenton's reagent. Conclusions: It is concluded that boeravinone G exhibits an extraordinary potent antioxidant activity (significant effect in the nanomolar range). The MAP kinase and NF-kB pathways seem to be involved in the antioxidant effect of boeravinone G. Boeravinone G might be considered as lead compound for the development of drugs potentially useful against those pathologies whose aetiology is related to ROS-mediated injuries

Environmental and Genetic Determinants of Colony Morphology in Yeast

Nutrient stresses trigger a variety of developmental switches in the budding yeast Saccharomyces cerevisiae. One of the least understood of such responses is the development of complex colony morphology, characterized by intricate, organized, and strain-specific patterns of colony growth and architecture. The genetic bases of this phenotype and the key environmental signals involved in its induction have heretofore remained poorly understood. By surveying multiple strain backgrounds and a large number of growth conditions, we show that limitation for fermentable carbon sources coupled with a rich nitrogen source is the primary trigger for the colony morphology response in budding yeast. Using knockout mutants and transposon-mediated mutagenesis, we demonstrate that two key signaling networks regulating this response are the filamentous growth MAP kinase cascade and the Ras-cAMP-PKA pathway. We further show synergistic epistasis between Rim15, a kinase involved in integration of nutrient signals, and other genes in these pathways. Ploidy, mating-type, and genotype-by-environment interactions also appear to play a role in the controlling colony morphology. Our study highlights the high degree of network reuse in this model eukaryote; yeast use the same core signaling pathways in multiple contexts to integrate information about environmental and physiological states and generate diverse developmental outputs

Quantitative differential proteomics of yeast extracellular matrix: there is more to it than meets the eye

Background: Saccharomyces cerevisiae multicellular communities are sustained by a scaffolding extracellular matrix, which provides spatial organization, and nutrient and water availability, and ensures group survival. According to this tissue-like biology, the yeast extracellular matrix (yECM) is analogous to the higher Eukaryotes counterpart for its polysaccharide and proteinaceous nature. Few works focused on yeast biofilms, identifying the flocculin Flo11 and several members of the HSP70 in the extracellular space. Molecular composition of the yECM, is therefore mostly unknown. The homologue of yeast Gup1 protein in high Eukaryotes (HHATL) acts as a regulator of Hedgehog signal secretion, therefore interfering in morphogenesis and cell-cell communication through the ECM, which mediates but is also regulated by this signalling pathway. In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation. Methods: S. cerevisiae W303-1A wt strain and gup1Δ mutant were used as previously described to generate biofilmlike mats in YPDa from which the yECM proteome was extracted. The proteome from extracellular medium from batch liquid growing cultures was used as control for yECM-only secreted proteins. Proteins were separated by SDS-PAGE and 2DE. Identification was performed by HPLC, LC-MS/MS and MALDI-TOF/TOF. The protein expression comparison between the two strains was done by DIGE, and analysed by DeCyder Extended Data Analysis that included Principal Component Analysis and Hierarchical Cluster Analysis. Results: The proteome of S. cerevisiae yECM from biofilm-like mats was purified and analysed by Nano LC-MS/MS, 2D Difference Gel Electrophoresis (DIGE), and MALDI-TOF/TOF. Two strains were compared, wild type and the mutant defective in GUP1. As controls for the identification of the yECM-only proteins, the proteome from liquid batch cultures was also identified. Proteins were grouped into distinct functional classes, mostly Metabolism, Protein Fate/Remodelling and Cell Rescue and Defence mechanisms, standing out the presence of heat shock chaperones, metalloproteinases, broad signalling cross-talkers and other putative signalling proteins. The data has been deposited to the ProteomeXchange with identifier PXD001133.Conclusions: yECM, as the mammalian counterpart, emerges as highly proteinaceous. As in higher Eukaryotes ECM, numerous proteins that could allow dynamic remodelling, and signalling events to occur in/and via yECM were identified. Importantly, large sets of enzymes encompassing full antagonistic metabolic pathways, suggest that mats develop into two metabolically distinct populations, suggesting that either extensive moonlighting or actual metabolism occurs extracellularly. The gup1Δ showed abnormally loose ECM texture. Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.This work was funded by the Marie Curie Initial Training Network GLYCOPHARM (PITN-GA-2012-317297), and by national funds from FCT I.P. through the strategic funding UID/BIA/04050/2013. Fábio Faria-Oliveira was supported by a PhD scholarship (SFRH/BD/45368/2008) from FCT (Fundação para a Ciência e a Tecnologia). We thank David Caceres and Montserrat MartinezGomariz from the Unidad de Proteómica, Universidad Complutense de Madrid – Parque Científico de Madrid, Spain for excellent technical assistance in the successful implementation of all proteomics procedures including peptide identification, and Joana Tulha from the CBMA, Universidade do Minho, Portugal, for helping with the SDS-PAGE experiments, and the tedious and laborious ECM extraction procedures. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium, via the PRIDE partner repository, with the dataset identifier PXD001133. We would like to thank the PRIDE team for all the help and support during the submission process.info:eu-repo/semantics/publishedVersio

Exploring the three PIPs and three TIPs of grapevine for transport of water and atypical substrates through heterologous expression in aqy-null yeast

Aquaporins are membrane channels that facilitate the transport of water and other small molecules across the cellular membranes. We examined the role of six aquaporins of Vitis vinifera (cv. Touriga nacional) in the transport of water and atypical substrates (other than water) in an aqy-null strain of Saccharomyces cerevisiae. Their functional characterization for water transport was performed by stopped-flow fluorescence spectroscopy. The evaluation of permeability coefficients (Pf) and activation energies (Ea) revealed that three aquaporins (VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2) are functional for water transport, while the other three (VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1) are non-functional. TIPs (VvTnTIP1;1 and VvTnTIP2;2) exhibited higher water permeability than VvTnPIP2;1. All functional aquaporins were found to be sensitive to HgCl2, since their water conductivity was reduced (24–38%) by the addition of 0.5 mM HgCl2. Expression of Vitis aquaporins caused different sensitive phenotypes to yeast strains when grown under hyperosmotic stress generated by KCl or sorbitol. Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance. Their sequence analyses revealed the presence of signature sequences for transport of ammonia, boron, CO2, H2O2 and urea. The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H2O2, suggesting the contribution of Vitis aquaporins in the transport of these speciesinfo:eu-repo/semantics/publishedVersio