Genome Sequences of Burkholderia sp. Strains CCGE1002 and H160, Isolated from Legume Nodules in Mexico and Brazil

The genome sequences of Burkholderia sp. strains CCGE1002 from Mexico and H160 from Brazil, isolated from legume nodules, are reported. Their gene contents in relation to plant-microbe interactions and xenobiotic degradation are discussed.     

Influence of Soil Physical Properties on Plants of the Mussununga Ecosystem, Brazil

Distribution ranges of plant species are related to physical variables of ecosystems that limit plant growth. Therefore, each plant species response to physical factors builds up the functional diversity of an ecosystem. The higher the species richness of an ecosystem, the larger the probability of maintaining functions and the higher the potential number of plant functional groups (FGs). Thus, the richness potentially increases the number of functions of the highly diverse Atlantic Rainforest domain in Brazil. Severe plant growth limitations caused by stress, however, decrease species richness. In the Spodosols of the Mussununga, an associated ecosystem of Atlantic Rainforest, the percentage of fine sand is directly related to water retention. Moreover, the depth of the cementation layer in the Mussununga??s sandy soil is a physical factor that can affect the plants?? stress gradients. When a shallow cementation layer depth is combined with low water retention in soils and with low fine sand percentage, the double stresses of flooding in the rainy season and water scarcity in the dry season result. This study aimed to identify FGs among Mussununga plant species responding to water stress gradients of soil and to verify the effects of the gradients on plant species richness of the Mussununga. A canonical correspondence analysis (CCA) of species abundance and soil texture variables was performed on 18 plots in six physiognomies of the Mussununga. Species richness rarefactions were calculated for each vegetation form to compare diversity. The two main axes of the CCA showed two FGs responding to soil texture and cementation layer depth: stress tolerator species and mesic species. Physical variables affect plant diversity, with species richness rising as the fine sand proportion also rises in the Mussununga. The effect of the cementation layer is not significantly related to species richness variation.     

DNA methylation-independent reversion of gemcitabine resistance by hydralazine in cervical cancer cells

Background

Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells.

Methodology/Principal Findings

The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity.

Conclusions/Significance

Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.     

The oxygen concentration in cultures modulates protein expression and enzymatic antioxidant responses in Metarhizium lepidiotae conidia

Conidia from Metarhizium spp. are used for integrated pest control; however, environmental factors diminish the effectivity of these programs. Several approaches tried to improve conidia resistance to overcome this limitation, although little is known about the mechanisms involved in this effect. Here we measured the activity of antioxidant enzymes and conidia virulence, comparing the proteomic profiles of Metarhiziumlepidiotae CP-OAX conidia produced under normal (21% O₂) and high oxygen atmospheres (pulses with 30% O₂). We detected a higher virulence against Tenebrio molitor larvae, in addition to an increase in ultraviolet light tolerance in conidia produced under 30% O₂, which correlates with increased glutathione reductase activity. Two-dimensional gel electrophoresis (2D SDS–PAGE) of proteins extracted in conidia harvested from both experimental conditions revealed a group of proteins that was observed only in conidia from oxidant atmospheres. Some of those proteins were directly involved in oxidative stress responses, whereas others were involved in conidial virulence, thermo-tolerance, and the central metabolism. Thus, a high atmospheric oxygen concentration (30%) activates antioxidant defence and general stress response mechanisms involved in conidia resistance to adverse environmental factors, which can ultimately translate into higher effectivity for the use of entomopathogenic fungi conidia in pest control.     

Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells

Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.     

Search for substrate-based inhibitors fitting the S2' space of malarial aspartic protease plasmepsin II.

Plasmepsin (Plm) has been identified as an important target for the development of new antimalarial drugs, since its inhibition leads to the starvation of Plasmodium falciparum. A series of substrate-based dipeptide-type Plm II inhibitors containing the hydroxymethylcarbonyl isostere as a transition-state mimic were synthesized. The general design principle was provision of a conformationally restrained hydroxyl group (corresponding to the set residue at the P2' position in native substrates) and a bulky unit to fit the S2' pocket.     

Isothermal titration calorimetry to determine association constants for high-affinity ligands

An important goal in drug development is to engineer inhibitors and ligands that have high binding affinities for their target molecules. In optimizing these interactions, the precise determination of the binding affinity becomes progressively difficult once it approaches and surpasses the nanomolar level. Isothermal titration calorimetry (ITC) can be used to determine the complete binding thermodynamics of a ligand down to the picomolar range by using an experimental mode called displacement titration. In a displacement titration, the association constant of a high-affinity ligand that cannot be measured directly is artificially lowered to a measurable level by premixing the protein with a weaker competitive ligand. To perform this protocol, two titrations must be carried out: a direct titration of the weak ligand to the target macromolecule and a displacement titration of the high-affinity ligand to the weak ligand-target macromolecule complex. This protocol takes approximately 5 h.     

cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells

We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces cholesterol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol transport in isolated mitochondria from nonstimulated cells. The effect of arachidonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accumulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regulation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role.     

Small-scale disturbances spread along trophic chains: leaf-cutting ant nests, plants, aphids, and tending ants

Small-scale disturbances caused by animals often modify soil resource availability and may also affect plant attributes. Changes in the phenotype of plants growing on disturbed, nutrient-enriched microsites may influence the distribution and abundance of associated insects. We evaluated how the high nutrient availability generated by leaf-cutting ant nests in a Patagonian desert steppe may spread along the trophic chain, affecting the phenotype of two thistle species, the abundance of a specialist aphid and the composition of the associated assemblage of tending ants. Plants of the thistle species Carduus nutans and Onopordum acanthium growing in piles of waste material generated by leaf-cutting ant nests (i.e., refuse dumps) had more leaves, inflorescences and higher foliar nitrogen content than those in non-nest soils. Overall, plants in refuse dumps showed higher abundance of aphids than plants in non-nest soils, and aphid colonies were of greater size on O. acanthium plants than on C. nutans plants. However, only C. nutans plants showed an increase in aphid abundance when growing on refuse dumps. This resulted in a similar aphid load in both thistle species when growing on refuse dumps. Accordingly, only C. nutans showed an increase in the number of ant species attending aphids when growing on refuse dumps. The increase of soil fertility generated by leaf-cutting ant nests can affect aphid abundance and their tending ant assemblage through its effect on plant size and quality. However, the propagation of small-scale soil disturbances through the trophic chain may depend on the identity of the species involved.