Methylation patterns revealed by MSAP profiling in genetically stable somatic embryogenic cultures of Ocotea catharinensis (Lauraceae)

Ocotea catharinensis is a rare tree species indigenous to the Atlantic rainforest of South America. In spite of its value as a hardwood species, it is in danger of extinction. The species erratically produces seeds showing irregular flowering and slow growth. Therefore, plants are not easily replaced. Tissue culture-based techniques are commonly used for obtaining living material for tree propagation and in vitro preservation. Therefore, a high-frequency somatic embryogenic system was developed for the species. In the present work, the genetic fidelity of cell aggregates and somatic embryos at various stages of in vitro development of O. catharinensis was investigated using RAPD and AFLP markers. Both analyses confirmed the absence of genetic variation in all developmental stages of O. catharinensis embryogenic cultures, verifying that the in vitro system is genetically stable. The cultures were also analyzed for their methylation profiles at 5`-CCGG-3` sites by identifying methylation-sensitive amplification polymorphisms. Some of these markers differentiated cell aggregates from embryo bodies. The sequencing of ten MSAP markers revealed that four sequences showed significant similarity to genes encoding plant proteins. Particularly, the predicted amino acid sequence of the fragment designated as OcEaggHMttc155 was similar to the enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO), which is involved in the biosynthesis of ethylene, and its expression was reported to occur from the beginning to the intermediate stages of plant embryo development. Here, we suggest that this enzyme is possibly involved in the control of the earliest stages of somatic embryogenesis of O. catharinensis, and an approach to study ACO expression during somatic embryogenesis is proposed.

Biochemical responses of the ethylene-insensitive Never ripe tomato mutant subjected to cadmium and sodium stresses

In order to further address the known interaction between ethylene and components of the oxidative system, we have used the ethylene-insensitive Never ripe (Nr) tomato (Solanum lycopersicum L) mutant, which blocks ethylene responses. The mutant was compared to the control Micro-Tom (MT) cultivar subjected to two stressful situations: 100 mM NaCl and 0.5 mM CdCl(2). Leaf chlorophyll, lipid peroxidation and antioxidant enzyme activities in roots, leaves and fruits, and Na and Cd accumulation in tissues were determined. Although we verified a similar growth pattern and Na and Cd accumulation for MT and Nr, the mutant exhibited reduced leaf chlorophyll degradation following stress. In roots and leaves, the patterns of catalase (CAT), glutathione reductase (GR), ascorbate peroxidase (APX), guaiacol peroxidase (GPOX), superoxide dismutase (SOD) enzyme activity as well as malondialdehyde (MDA) and hydrogen peroxide (H(2)O(2)) production under the stressful conditions tested were very similar between MT and Nr mutant. However, Nr fruits showed increased H(2)O(2) production, reduced and enhanced APX activity in NaCl and CdCl(2), respectively, and enhanced GPOX in NaCl. Moreover, through non-denaturing PAGE, a similar reduction of SOD I band intensity in both, control MT and Nr mutant, treated with NaCl was observed. In leaves and fruits, a similar SOD activity pattern was observed for all periods, genotypes and treatments. Overall the results indicate that the ethylene signaling associated with NR receptor can modulate the biochemical pathways of oxidative stress in a tissue dependent manner, and that this signaling may be different following Na and Cd exposure. (C) 2011 Elsevier B.V. All rights reserved.

Transgenic tobacco revealing altered bacterial diversity in the rhizosphere during early plant development

The rhizosphere constitutes a complex niche that may be exploited by a wide variety of bacteria. Bacterium-plant interactions in this niche can be influenced by factors such as the expression of heterologous genes in the plant. The objective of this work was to describe the bacterial communities associated with the rhizosphere and rhizoplane regions of tobacco plants, and to compare communities from transgenic tobacco lines (CAB1, CAB2 and TRP) with those found in wild-type (WT) plants. Samples were collected at two stages of plant development, the vegetative and flowering stages (1 and 3 months after germination). The diversity of the culturable microbial community was assessed by isolation and further characterization of isolates by amplified ribosomal RNA gene restriction analysis (ARDRA) and 16S rRNA sequencing. These analyses revealed the presence of fairly common rhizosphere organisms with the main groups Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacilli. Analysis of the total bacterial communities using PCR-DGGE (denaturing gradient gel electrophoresis) revealed that shifts in bacterial communities occurred during early plant development, but the reestablishment of original community structure was observed over time. The effects were smaller in rhizosphere than in rhizoplane samples, where selection of specific bacterial groups by the different plant lines was demonstrated. Clustering patterns and principal components analysis (PCA) were used to distinguish the plant lines according to the fingerprint of their associated bacterial communities. Bands differentially detected in plant lines were found to be affiliated with the genera Pantoea, Bacillus and Burkholderia in WT, CAB and TRP plants, respectively. The data revealed that, although rhizosphere/rhizoplane microbial communities can be affected by the cultivation of transgenic plants, soil resilience may be able to restore the original bacterial diversity after one cycle of plant cultivation.

Spatial patterns of soil attributes and components in a mangrove system in Southeast Brazil (Sao Paulo)

Purpose Among environmental factors governing innumerous processes that are active in estuarine environments, those of edaphic character have received special attention in recent studies. With the objectives of determining the spatial patterns of soil attributes and components across different mangrove forest landscapes and obtaining additional information on the cause-effect relationships between these variables and position within the estuary, we analyzed several soil attributes in 31 mangrove soil profiles from the state of So Paulo (Guaruja, Brazil). Materials and methods Soil samples were collected at low tide along two transects within the CrumahA(0) mangrove forest. Samples were analyzed to determine pH, Eh, salinity, and the percentages of sand, silt, clay, total organic carbon (TOC), and total S. Mineralogy of the clay fraction (< 2 mm) was also studied by X-ray diffraction analysis, and partitioning of solid-phase Fe was performed by sequential extraction. Results and discussion The results obtained indicate important differences in soil composition at different depths and landscape positions, causing variations in physicochemical parameters, clay mineralogy, TOC contents, and iron geochemistry. The results also indicate that physicochemical conditions may vary in terms of different local microtopographies. Soil salinity was determined by relative position in relation to flood tide and transition areas with highlands. The proportions of TOC and total S are conditioned by the sedimentation of organic matter derived from vegetation and by the prevailing redox conditions, which clearly favored intense sulfate reduction in the soils (similar to 80% of the total Fe is Fe-pyrite). Particle-size distribution is conditioned by erosive/deposition processes (present and past) and probably by the positioning of ancient and reworked sandy ridges. The existing physicochemical conditions appear to contribute to the synthesis (smectite) and transformation (kaolinite) of clay minerals. Conclusions The results demonstrate that the position of soils in the estuary greatly affects soil attributes. Differences occur even at small scales (meters), indicating that both edaphic (soil classification, soil mineralogy, and soil genesis) and environmental (contamination and carbon stock) studies should take such variability into account.

Out of the Andes: patterns of diversification in clearwing butterflies

Global biodiversity peaks in the tropical forests of the Andes, a striking geological feature that has likely been instrumental in generating biodiversity by providing opportunities for both vicariant and ecological speciation. However, the role of these mountains in the diversification of insects, which dominate biodiversity, has been poorly explored using phylogenetic methods. Here we study the role of the Andes in the evolution of a diverse Neotropical insect group, the clearwing butterflies. We used dated species-level phylogenies to investigate the time course of speciation and to infer ancestral elevation ranges for two diverse genera. We show that both genera likely originated at middle elevations in the Andes in the Middle Miocene, contrasting with most published results in vertebrates that point to a lowland origin. Although we detected a signature of vicariance caused by the uplift of the Andes at the Miocene-Pliocene boundary, most sister species were parapatric without any obvious vicariant barrier. Combined with an overall decelerating speciation rate, these results suggest an important role for ecological speciation and adaptive radiation, rather than simple vicariance.

Interaction between forest fragments and adjacent coffee and pasture agroecosystems: responses of the ant communities (Hymenoptera, Formicidae)

The responses of the ant community to environmental change, from forest fragment to agroecosystems (coffee or pasture) were evaluated in the south of the state of Minas Gerais, Brazil. In this paper we analized the interactions between forest and the two most typical agroecosystem from southest Brazil: sun-growing coffee plantation and introduced pasture. We sampled the ant community from five of each agroecosystems, inside the adjacent forest fragment, and on the edge between them. In each site we removed the litter from fifteen 1m(2) plots and extracted the ants using a Winkler extractor. A total of 165 ant species, distributed in 48 genera and 10 subfamilies were recorded. The coffee plantation presented the lowest abundance and estimated species richness. The causes of the changes observed among the areas are discussed.

Interaction Between Polar Components and the Degree of Unsaturation of Fatty Acids on the Oxidative Stability of Emulsions

Minor components (polar components) and the degree of unsaturation of the fatty acids are the main factors responsible for the oxidative stability of bulk oils and emulsions. The isolated effects of these two factors and their interaction were evaluated in oil-in-water emulsions stored at 32 A degrees C. Samples of coconut, olive, soybean, linseed and fish oils, both full and stripped of their polar components, were used to prepare the emulsions (1% w/w). The maximum concentration of hydroperoxide (LOOH(max)) and the rate of formation of hydroperoxides (mu mol L(-1) h(-1)) were used to measure the primary products. Hexanal, propanal and malondialdehyde were used to determine the secondary products of the oxidized emulsions containing polyunsaturated fatty acids. LOOH(max) varied from 0.16 to 12.75 mmol/kg among the samples. The interaction between the polar components and the degree of unsaturation of the fatty acids was significant (p < 0.001) when the hydroperoxides were evaluated. In general, the degree of unsaturation (beta(1)) and the absence of polar components (beta(2)), respectively, represented 30 and 20% of the contribution to increase the mean oxidation, with the interaction (beta(12)) contribution being more sensitive to the rate of formation of hydroperoxides (16%) than to the LOOH(max) (5%). The significance of this interaction suggests that both strategies present synergism and should be applied to improve the oxidative stability of food emulsions.

Enteroinvasive Escherichia coli vs. Shigella flexneri : how different patterns of gene expression affect virulence

Important features of the enteroinvasive Escherichia coli (EIEC) phenotype and gene expression likely to confer EIEC with a lower ability to cause disease than Shigella flexneri were described here for the first time. To confirm the lower pathogenicity of EIEC, we have analyzed the keratoconjunctivitis developed in guinea-pigs with EIEC or S. flexneri. Shigella flexneri induced a more pronounced proinflammatory response, whereas EIEC induced a mild form of the disease. EIEC showed a significantly less efficient cell-to-cell Caco-2 dissemination when compared with S. flexneri. Plaques formed by EIEC during intercellular spreading were four times smaller than those formed by S. flexneri. At the molecular level, the lower expression of virulence genes by EIEC during infection of Caco-2 cells highlighted the importance of effective gene transcription for bacterial pathogenicity.

Cell surface expression of adhesins for fibronectin correlates with virulence in Sporothrix schenckii

The virulence of four Sporothrix schenckii isolates was compared in a murine model of sporotrichosis, together with the protein pattern of the yeast cell surface and the capacity to bind the extracellular matrix protein fibronectin. Virulence was determined by the mortality rate, fungal burden and histopathology. Two clinical isolates were more virulent for C57BL/6 mice, but no direct correlation was seen between virulence and the clinical or environmental origin of the isolates. The lowest virulence was observed for an isolate recovered from a patient with meningeal sporotrichosis. Although all isolates could effectively disseminate, the dissemination patterns were not similar. Using flow cytometry analysis, we investigated the interaction of all the strains with fibronectin, and showed that the binding capacity correlated with virulence. Western blot analysis of S. schenckii cell wall extracts revealed positive bands for fibronectin in the range of 3792 kDa. The 70 kDa adhesin was also recognized by a protective monoclonal antibody raised against a gp70 antigen of S. schenckii (mAb P6E7). Confocal microscopy confirmed the co-localization of fibronectin and mAb P6E7 on the yeast cell surface. To our knowledge, this is the first report identifying adhesins for fibronectin on the surface of this human pathogen.

Study of the interaction between hydroxymethylnitrofurazone and 2-hydroxypropyl-beta-cyclodextrin

Chagas disease is a serious health problem in Latin America. Hidroxymethylnitrofurazone (NFOH) is a nitrofurazone prodrug more active than nitrofurazone against Trypanosoma cruzi. However, NFOH presents low aqueous solubility, high photodecomposition and high toxicity. The present work is focused on the characterization of an inclusion complex of NFOH in 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD). The complexation with HP-beta-CD was investigated using reversed-phase liquid chromatography, solubility isotherms and nuclear magnetic resonance. The retention behavior was analyzed on a reversed-phase C-18 column, using acetonitrile-water (20/80, v/v) as the mobile phase, in which HP-beta-CD was incorporated as a mobile phase additive. The decrease in the retention times with increasing concentrations of HP-beta-CD enables the determination of the apparent stability constant of the complex (K = 6.2 +/- 0.3 M-1) by HPLC. The solubility isotherm was studied and the value for the apparent stability constant (K = 7.9 +/- 0.2 M-1) was calculated. The application of continuous variation method indicated the presence of a complex with 1:1 NFOH:HP-beta-CD stoichiometry. The photostability study showed that the formation of an inclusion complex had a destabilizing effect on the photodecomposition of NFOH when compared to that of the ""free"" molecule in solution. The mobility investigation (by NMR longitudinal relaxation time) gives information about the complexation of NFOH with HP-beta-CD. In preliminary toxicity studies, cell viability tests revealed that inclusion complexes were able to decrease the toxic effect (p < 0.01) caused by NFOH. (c) 2008 Elsevier B.V. All rights reserved.

Generation and Analysis of Interaction Energy Maps of p-Substituted Benzoic Acid [(5-Nitro-thiophen-2-yl)-methylene]-Hydrazides Active against MRSA

In this preliminary study eighteen p-substituted benzoic acid [(5-nitro-thiophen-2-yl)-methylene]-hydrazides with antimicrobial activity were evaluated against multidrug-resistant Staphylococcus aureus, correlating the three-dimensional characteristics of the ligands with their respective bioactivities. The computer programs Sybyl and CORINA were used, respectively, for the design and three-dimensional conversion of the ligands. Molecular interaction fields were calculated using GRID program. Calculations using Volsurf resulted in a statistically consistent model with 48 structural descriptors showing that hydrophobicity is a fundamental property in the analyzed biological response.

Expression patterns of cell wall-modifying genes from banana during fruit ripening and in relationship with finger drop

Few molecular studies have been devoted to the finger drop process that occurs during banana fruit ripening. Recent studies revealed the involvement of changes in the properties of cell wall polysaccharides in the pedicel rupture area. In this study, the expression of cell-wall modifying genes was monitored in peel tissue during post-harvest ripening of Cavendish banana fruit, at median area (control zone) and compared with that in the pedicel rupture area (drop zone). To this end, three pectin methylesterase (PME) and seven xyloglucan endotransglycosylase/hydrolase (XTH) genes were isolated. The accumulation of their mRNAs and those of polygalaturonase, expansin, and pectate lyase genes already isolated from banana were examined. During post-harvest ripening, transcripts of all genes were detected in both zones, but accumulated differentially. MaPME1, MaPG1, and MaXTH4 mRNA levels did not change in either zone. Levels of MaPME3 and MaPG3 mRNAs increased greatly only in the control zone and at the late ripening stages. For other genes, the main molecular changes occurred 1-4 d after ripening induction. MaPME2, MaPEL1, MaPEL2, MaPG4, MaXTH6, MaXTH8, MaXTH9, MaEXP1, MaEXP4, and MaEXP5 accumulated highly in the drop zone, contrary to MaXTH3 and MaXTH5, and MaEXP2 throughout ripening. For MaPG2, MaXET1, and MaXET2 genes, high accumulation in the drop zone was transient. The transcriptional data obtained from all genes examined suggested that finger drop and peel softening involved similar mechanisms. These findings also led to the proposal of a sequence of molecular events leading to finger drop and to suggest some candidates.

Cubebin and derivatives as inhibitors of mitochondrial complex I. Proposed interaction with subunit B8

The effects on mitochondrial respiration and complex I NADH oxidase activity of cubebin and derivatives were evaluated. The compounds inhibited the state 3 glutamate/malate-supported respiration of hamster liver mitochondria with IC50 values ranging from 12.16 to 83.96M. NADH oxidase reaction was evaluated in submitochondrial particles. The compounds also inhibited this activity, showing the same order of potency observed for effects on state 3 respiration, as well as a tendency towards a non-competitive type of inhibition (KI values ranging from 0.62 to 16.1M). A potential binding mode of these compounds with complex I subunit B8, assessed by docking calculations, is proposed.

Molecular dynamics, density functional, ADMET predictions, virtual screening, and molecular interaction field studies for identification and evaluation of novel potential CDK2 inhibitors in cancer therapy

In this work, we have used molecular dynamics, density functional theory, virtual screening, ADMET predictions, and molecular interaction field studies to design and propose eight novel potential inhibitors of CDK2. The eight molecules proposed showed interesting structural characteristics that are required for inhibiting the CDK2 activity and show potential as drug candidates for the treatment of cancer. The parameters related to the Rule of Five were calculated, and only one of the molecules violated more than one parameter. One of the proposals and one of the drug-like compounds selected by virtual screening indicated to be promising candidates for CDK2-based cancer therapy.

Use of virtual screening, flexible docking, and molecular interaction fields to design novel HMG-CoA reductase inhibitors for the treatment of hypercholesterolemia

Dietary changes associated with drug therapy can reduce high serum cholesterol levels and dramatically decrease the risk of coronary artery disease, stroke, and overall mortality. Statins are hypolipemic drugs that are effective in the reduction of cholesterol serum levels, attenuating cholesterol synthesis in liver by competitive inhibition regarding the substrate or molecular target HMG-CoA reductase. We have herewith used computer-aided molecular design tools, i.e., flexible docking, virtual screening in large data bases, molecular interaction fields to propose novel potential HMG-CoA reductase inhibitors that are promising for the treatment of hypercholesterolemia.