Bacteria associated with the pinewood nematode <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis> collected in Portugal

In this study, we report on the bacterial community associated with the pinewood nematode Bursaphelenchus xylophilus from symptomatic pine wilted trees, as well as from long-term preserved B. xylophilus laboratory collection specimens, emphasizing the close bacteria–nematode associations that may contribute to pine wilt disease development.     

Localization and function of adenosine receptor subtypes at the longitudinal muscle--myenteric plexus of the rat ileum

Adenosine plays a dual role on acetylcholine (ACh) release from myenteric motoneurons via the activation of high-affinity inhibitory A₁ and facilitatory A_2A receptors. The therapeutic potential of adenosine-related compounds for controlling intestinal motility and inflammation, prompted us to investigate further the role of low-affinity adenosine receptors, A_2B and A₃, on electrically-evoked (5 Hz, 200 pulses) [³H]ACh release from myenteric neurons. Immunolocalization studies showed that A_2B receptors exhibit a pattern of distribution similar to the glial cell marker, GFAP. Regarding A₁ and A₃ receptors, they are mainly distributed to cell bodies of ganglionic myenteric neurons, whereas A_2A receptors are localized predominantly on cholinergic nerve terminals. Using selective antagonists (DPCPX, ZM241385 and MRS1191), data indicate that modulation of evoked [³H]ACh release is balanced through tonic activation of inhibitory (A₁) and facilitatory (A_2A and A₃) receptors by endogenous adenosine. The selective A_2B receptor antagonist, PSB603, alone was devoid of effect and failed to modify the inhibitory effect of NECA. The A₃ receptor agonist, 2-Cl-IB MECA (1–10 nM), concentration-dependently increased the release of [³H]ACh. The effect of 2-Cl-IB MECA was attenuated by MRS1191 and by ZM241385, which selectively block respectively A₃ and A_2A receptors. In contrast to 2-Cl-IB MECA, activation of A_2A receptors with CGS21680C attenuated nicotinic facilitation of ACh release induced by focal depolarization of myenteric nerve terminals in the presence of tetrodotoxin. Tandem localization of excitatory A₃ and A_2A receptors along myenteric neurons explains why stimulation of A₃ receptors (with 2-Cl-IB MECA) on nerve cell bodies acts cooperatively with prejunctional facilitatory A_2A receptors to up-regulate acetylcholine release. The results presented herein consolidate and expand the current understanding of adenosine receptor distribution and function in the myenteric plexus of the rat ileum, and should be taken into consideration for data interpretation regarding the pathophysiological implications of adenosine on intestinal motility disorders.     

Dengue virus type 4 phylogenetics in Brazil 2011: looking beyond the veil

Dengue Fever and Dengue Hemorrhagic Fever are diseases affecting approximately 100 million people/year and are a major concern in developing countries. In the present study, the phylogenetic relationship of six strains of the first autochthonous cases of DENV-4 infection occurred in Sao Paulo State, Parana State and Rio Grande do Sul State, Brazil, 2011 were studied. Nucleotide sequences of the envelope gene were determined and compared with sequences representative of the genotypes I, II, III and Sylvatic for DEN4 retrieved from GenBank. We employed a Bayesian phylogenetic approach to reconstruct the phylogenetic relationships of Brazilian DENV-4 and we estimated evolutionary rates and dates of divergence for DENV-4 found in Brazil in 2011. All samples sequenced in this study were located in Genotype II. The studied strains are monophyletic and our data suggest that they have been evolving separately for at least 4 to 6 years. Our data suggest that the virus might have been present in the region for some time, without being noticed by Health Surveillance Services due to a low level of circulation and a higher prevalence of DENV-1 and DENV- 2.     

An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli

Background

Membrane proteins play a key role in many fundamental cellular processes such as transport of nutrients, sensing of environmental signals and energy transduction, and account for over 50% of all known drug targets. Despite their importance, structural and functional characterisation of membrane proteins still remains a challenge, partially due to the difficulties in recombinant expression and purification. Therefore the need for development of efficient methods for heterologous production is essential.

Methodology/Principal Findings

Fifteen integral membrane transport proteins from Archaea were selected as test targets, chosen to represent two superfamilies widespread in all organisms known as the Major Facilitator Superfamily (MFS) and the 5-Helix Inverted Repeat Transporter superfamily (5HIRT). These proteins typically have eleven to twelve predicted transmembrane helices and are putative transporters for sugar, metabolite, nucleobase, vitamin or neurotransmitter. They include a wide range of examples from the following families: Metabolite-H⁺-symporter; Sugar Porter; Nucleobase-Cation-Symporter-1; Nucleobase-Cation-Symporter-2; and neurotransmitter-sodium-symporter. Overproduction of transporters was evaluated with three vectors (pTTQ18, pET52b, pWarf) and two Escherichia coli strains (BL21 Star and C43 (DE3)). Thirteen transporter genes were successfully expressed; only two did not express in any of the tested vector-strain combinations. Initial trials showed that seven transporters could be purified and six of these yielded quantities of ≥ 0.4 mg per litre suitable for functional and structural studies. Size-exclusion chromatography confirmed that two purified transporters were almost homogeneous while four others were shown to be non-aggregating, indicating that they are ready for up-scale production and crystallisation trials.

Conclusions/Significance

Here, we describe an efficient strategy for heterologous production of membrane transport proteins in E. coli. Small-volume cultures (10 mL) produced sufficient amount of proteins to assess their purity and aggregation state. The methods described in this work are simple to implement and can be easily applied to many more membrane proteins.     

Mycobacterium tuberculosis Strains Are Differentially Recognized by TLRs with an Impact on the Immune Response

Tuberculosis associates with a wide spectrum of disease outcomes. The Beijing (Bj) lineage of Mycobacterium tuberculosis (Mtb) is suggested to be more virulent than other Mtb lineages and prone to elicit non-protective immune responses. However, highly heterogeneous immune responses were reported upon infection of innate immune cells with Bj strains or stimulation with their glycolipids. Using both in vitro and in vivo mouse models of infection, we here report that the molecular mechanism for this heterogeneity may be related to distinct TLR activations. Among this Mtb lineage, we found strains that preferentially activate TLR2, and others that also activate TLR4. Recognition of Mtb strains by TLR4 resulted in a distinct cytokine profile in vitro and in vivo, with specific production of type I IFN. We also uncover a novel protective role for TLR4 activation in vivo. Thus, our findings contribute to the knowledge of the molecular basis underlying how host innate immune cells handle different Mtb strains, in particular the intricate host-pathogen interaction with strains of the Mtb Bj lineage.     

Antitumor Activity of Hierridin B,a Cyanobacterial Secondary Metabolite Found in both Filamentous and Unicellular Marine Strains

Cyanobacteria are widely recognized as a valuable source of bioactive metabolites. The majority of such compounds have been isolated from so-called complex cyanobacteria, such as filamentous or colonial forms, which usually display a larger number of biosynthetic gene clusters in their genomes, when compared to free-living unicellular forms. Nevertheless, picocyanobacteria are also known to have potential to produce bioactive natural products. Here, we report the isolation of hierridin B from the marine picocyanobacterium Cyanobium sp. LEGE 06113. This compound had previously been isolated from the filamentous epiphytic cyanobacterium Phormidium ectocarpi SAG 60.90, and had been shown to possess antiplasmodial activity. A phylogenetic analysis of the 16S rRNA gene from both strains confirmed that these cyanobacteria derive from different evolutionary lineages. We further investigated the biological activity of hierridin B, and tested its cytotoxicity towards a panel of human cancer cell lines; it showed selective cytotoxicity towards HT-29 colon adenocarcinoma cells.     

Global environmental changes: setting priorities for Latin American coastal habitats

As the effects of the Global Climate Changes on the costal regions of Central and South Americas advance, there is proportionally little research being made to understand such impacts. This commentary puts forward a series of propositions of strategies to improve performance of Central and South American science and policy making in order to cope with the future impacts of the Global Climate Changes in their coastal habitats.     

A substrate translocation trajectory in a cytoplasm-facing topological model of the monocarboxylate/H⁺ symporter Jen1p

Previous mutational analysis of Jen1p, a Saccharomyces cerevisiae monocarboxylate/H⁺ symporter of the Major Facilitator Superfamily, has suggested that the consensus sequence ³⁷⁹NXX[S/T]HX[S/T]QD³⁸⁷ in transmembrane segment VII (TMS‐VII) is part of the substrate translocation pathway. Here, we rationally design, analyse and show that several novel mutations in TMS‐V and TMS‐XI directly modify Jen1p function. Among the residues studied, F270 (TMS‐V) and Q498 (TMS‐XI) are critical specificity determinants for the distinction of mono‐ from dicarboxylates, and N501 (TMS‐XI) is a critical residue for function. Using a model created on the basis of Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS‐VII and TMS‐XI (N379, H383, D387, Q498, N501) are perfectly aligned in an imaginary axis that lies parallel to the protein pore. This model and subsequent mutational analysis further reveal that an additional polar residue facing the pore, R188 (TMS‐II), is irreplaceable for function. Our model also justifies the role of F270 and Q498 in substrate specificity. Finally, docking calculations reveal a ‘trajectory‐like’ substrate displacement within the Jen1p pore, where R188 plays a major dynamic role mediating the orderly relocation of the substrate by subsequent H‐bond interactions involving itself and residues H383, N501 and Q498.     

Activated carbons from sisal waste by chemical activation with K₂CO₃: kinetics of paracetamol and ibuprofen removal from aqueous solution

Sisal waste was used as precursor to prepare carbons by chemical activation. The influence of the K₂CO₃ amount and activation temperature on the materials textural properties were studied through N₂ and CO₂ adsorption assays. As the severity of the treatment increases there is a development of supermicropores, and the micropore size distribution changes from mono to bimodal. A carbon with an apparent surface area of 1038 m² g⁻¹ and pore volume of 0.49 cm³ g⁻¹ was obtained. TPD results showed the incidence in acidic type groups although the pH_PZC reveals an almost neutral character of the surface. Adsorption kinetic data of ibuprofen and paracetamol show that the processes obey to a pseudo-second order kinetic equation. Regarding the removal efficiency the prepared samples attained values comparable to a commercial carbon (>65%), revealing that chemical activation of sisal wastes with K₂CO₃ allows obtaining samples suitable for pharmaceutical compounds removal from liquid phase.     

Effect of Annexin A5 on CFTR: regulated traffic or scaffolding?

Previous studies have implicated annexins in regulating ion channels and in particular annexin A5 (AnxA5) in the traffic of the cystic fibrosis transmembrane conductance regulator (CFTR). In the present study, we further investigated the role of AnxA5 in regulating CFTR function and intracellular trafficking in both Xenopus oocytes and mammalian cells. Although we could confirm the previously reported CFTR/AnnxA5 interaction, we found that in oocytes AnxA5 inhibits CFTR-mediated whole-cell membrane conductance presumably by a mechanism independent of PDZ-binding domain at the C-terminus of CFTR but protein kinase C (PKC)-dependent and results from either endocytosis activation and/or exocytosis block. In contrast, in human cells, co-expression of AnxA5 augmented CFTR whole-cell currents, an effect that was independent of CFTR PDZ-binding domain. We conclude that annexin A5 has multiple effects on CFTR, so that the net effect observed is cell system-dependent. Nevertheless, both effects observed here are consistent with the described role of annexins forming scaffolding platforms at cell membranes, thus contributing to a decrease in their dynamics. Finally, we could not confirm that AnxA5 overexpression rescues traffic/function of the most frequent disease-causing mutant F508del-CFTR, thus concluding that AnxA5 is not a promising tool for correction of the F508del-CFTR defect.