14-3-3 proteins double the number of outward-rectifying K+ channels available for activation in tomato cells

Outward-rectifying K+ channels are modulated in response to environmental stimuli by a range of intracellular factors, such as cytoplasmic Ca2+, pH, kinases and phosphatases. Here we report that voltage-dependent outward-rectifying K+ channels in tomato cells are also targets for modulation by 14-3-3 proteins. In whole-cell patch-clamp experiments, recombinant 14-3-3 protein (tomato isoform TFT7) was introduced into tomato cell protoplasts via the patch pipette. As a result the steady-state outward K+ current increased twofold and this increase was not dependent upon the presence of cytoplasmic ATP. A phosphorylated peptide that contained a phosphorylated 14-3-3 target-binding motif (RSTS*TP), derived from nitrate reductase, blocked the effect of 14-3-3, thus showing the specific nature of 14-3-3 action. Kinetic parameters of the conductance, like (de)activation kinetics, voltage dependence of gating and activation potential, were not significantly different between control and 14-3-3 infused cells. Analysis of single-channel activity and whole-cell noise indicated that the single-channel conductance was not affected by 14-3-3 infusion. We conclude that 14-3-3 proteins recruit 'sleepy' channels into a voltage-activatable state. The molecular mechanism underlying the 1 : 1 ratio of constitutively active and 14-3-3 recruited channels is discussed in the light of known functions of 14-3-3 dimers.     

Longitudinal proxy measurements in multiple sclerosis: patient-proxy agreement on the impact of MS on daily life over a period of two years

Background  

The use of self-report measurements in clinical settings is increasing. However, in patients with limitations that interfere with reliable self-assessment such as cognitive impairment or mood disturbances, as may be the case in multiple sclerosis (MS), data collection might be problematic. In these situations, information obtained from proxy respondents (e.g. partners) may replace self-ratings. The aim of this study was to examine the value of proxy ratings at separate points in time and to assess patient-proxy agreement on possible changes in disease impact of MS.     

Direct enzymatic glucosylation of naringin in grapefruit juice by alpha-D-glucosidase from the marine mollusc Aplysia fasciata

The enzymatic glucosylations of naringin, performed using alpha-D-glucosidase, identified in the Mediterranean mollusc Aplysia fasciata is reported. The enzyme actively operates on maltose and has an interesting transglycosylation potential using this donor. We also investigated the use of this marine alpha-glucosidase for a food-compatible glucosylation of naringin to produce new enzymatically modified carbohydrate possessing naringin derivatives. The regioselective formations of the beta-gluco-C6 alpha-glucosyl derivative and of the corresponding isomaltosyl diglucoside of naringin were obtained in high yield and efficiency of reaction. Suspensions of naringin can be used up to approximately 90 mg/mL initial acceptor concentration. In different experiments it was demonstrated that the enzyme was still active after 48 h in presence of this high amount of acceptor and that one of the diasteromers of the naringin is preferred by the enzyme from A. fasciata during glucosylation/deglucosylation enzymatic steps. Finally, the feasibility of efficient naringin glucosylation in grapefruit juice was also demonstrated at optimal pH of the enzyme and low maltose concentrations.     

Interactions of Paracoccidioides brasiliensis with host cells: recent advances

Host-fungal interactions are inherently complex and dynamic. In order to identify new microbial targets and develop more effective anti-fungal therapies, it is important to understand the cellular and molecular mechanisms of disease. Paracoccidioidomycosis provokes a variety of clinical symptoms, and Paracoccidioides brasiliensis can reach many tissues, but primarily attacks the lungs. The ability of the pathogen to interact with the host surface structures is essential to further colonization, invasion, and growth. Epithelial cells may represent the first host barrier or the preferential site of entry of the fungus. For this reason, interactions between P. brasiliensis and Vero/A549 epithelial cells were evaluated, with an emphasis on the adherence, induction of cytoskeletal alterations, and differential signaling activity of the various surface molecules. The adhesion to and invasion of epithelial cells by P. brasiliensis may represent strategies employed to thwart the initial host immune response, and may help in the subsequent dissemination of the pathogen throughout the body.     

Design of a disulfide-less, pharmacologically inert, and chemically competent analog of maurocalcine for the efficient transport of impermeant compounds into cells

Maurocalcine is a 33-mer peptide initially isolated from the venom of a Tunisian scorpion. It has proved itself valuable as a pharmacological activator of the ryanodine receptor and has helped the understanding of the molecular basis underlying excitation-contraction coupling in skeletal muscles. Because of its positively charged nature, it is also an innovative vector for the cell penetration of various compounds. We report a novel maurocalcine analog with improved properties: (i) the complete loss of pharmacological activity, (ii) preservation of the potent ability to carry cargo molecules into cells, and (iii) coupling chemistries not affected by the presence of internal cysteine residues of maurocalcine. We did this by replacing the six internal cysteine residues of maurocalcine by isosteric 2-aminobutyric acid residues and by adding an additional N-terminal biotinylated lysine (for a proof of concept analog) or an N-terminal cysteine residue (for a chemically competent coupling analogue). Additional replacement of a glutamate residue by alanyl at position 12 further improves the potency of these analogues. Coupling to several cargo molecules or nanoparticles are presented to illustrate the cell penetration potency and usefulness of these pharmacologically inactive analogs.     

Competing modes of self-association in the regulatory domains of Bruton's tyrosine kinase: Intramolecular contact versus asymmetric homodimerization

A nuclear magnetic resonance (NMR) investigation of a fragment of the nonreceptor Tec family tyrosine kinase Btk has revealed an intricate set of coupled monomer-dimer equilibria. The Btk fragment studied contains two consecutive proline-rich motifs followed by a single Src homology 3 (SH3) domain. We provide evidence for an asymmetric homodimer in which the amino-terminal proline sequence of one monomer contacts the opposite SH3 binding pocket, whereas the carboxy-terminal proline sequence of the other monomer is engaged by the second SH3 domain across the dimer interface. We show that the asymmetric homodimer structure is mimicked by a heterodimer formed in an equimolar mixture of complimentary mutants: one carrying mutations in the amino-terminal proline stretch; the other, in the carboxy-terminal proline motif. Moreover, a monomeric species characterized by an intramolecular complex between the amino-terminal proline motif and the SH3 domain predominates at low concentration. Association constants were determined for each of the competing equilibria by NMR titration. The similarity of the determined K(a) values reveals a delicate balance between the alternative conformational states available to Btk. Thus, changes in the local concentration of Btk itself, or co-localization with exogenous signaling molecules that have high affinity for either proline sequence or the SH3 domain, can significantly alter species composition and regulate Btk kinase activity.     

Conformational changes in pediocin AcH upon vesicle binding and approximation of the membrane-bound structure in detergent micelles.

Pediocin AcH is a 44-residue antimicrobial peptide with bactericidal potency against Gram-positive bacteria such as Listeria. It belongs to a family of bacteriocins that, when membrane-associated, is predicted to contain beta-sheet and alpha-helical regions. All bacteriocins in this family have a conserved N-terminal disulfide bond. An additional C-terminal disulfide bond in pediocin AcH is thought to confer enhanced potency and broader specificity range against sensitive bacteria. The C-terminal disulfide bond may also affect the conformation of the C-terminus. The secondary structures of pediocin AcH in aqueous solution and vesicles from susceptible cells, as well as the ability of trifluoroethanol (TFE) and detergent systems to induce secondary structures like those induced in vesicles, were studied by circular dichroism (CD) spectroscopy. Like related peptides, pediocin AcH was highly unordered in aqueous solution, 56%. However, it also contained 20% beta-strand and 15% beta-turn structures. Upon complete binding to vesicles, 32% alpha-helical structure formed, the unordered structure decreased to 32%, and the beta-strand and beta-turn structures remained largely unchanged. Thus, a betaalpha domain structure formed in vesicles. The helical structure likely forces the C-terminal tail to loop back on the helix so that the C24-C44 disulfide bond can form. Detergent micelles were superior to TFE in their ability to induce secondary structural fractions in pediocin AcH comparable to those observed in vesicles. This demonstrates the importance of a hydrocarbon-water interface to pediocin AcH structure induction and suggests that it is preferable to use detergent micelles as solvents in NMR studies of pediocin AcH structure.     

Benthic freshwater nematode community dynamics under conditions of Tilapia aquaculture in Egypt

Studies of the influence of fish aquaculture on benthic freshwater nematode assemblages are scarce, but could provide a way of gauging environmental effects. The abundance and diversity of nematode assemblages in response to Oreochromis niloticus aquaculture were investigated in Kafr El-Sheikh Governorate, Egypt, from July to November 2014 under conditions of irrigation (reference), fish farm pond with high Tilapia density, and fish farm pond effluent canal without fish. The nematode genera Adoncholaimus, Punctodora, Labronema, Oncholaimus and Odontolaimus were present at all sites. Environmental factors were not related to nematode distribution patterns. Tilapia predation and/or disturbance may explain reduced nematode abundance, especially of the largest genera, Adoncholaimus, Punctodora and Labronema at the fish farm site. The absence of fish from the drainage site allowed intergeneric nematode competitive exclusion, benefitting the largest nematodes and reducing diversity indices.     

Chemical characterization and antifungal activity of essential oil of capitula from wild Indian Tagetes patula L.

Summary. The essential oil extracted by steam distillation from the capitula of Indian Tagetes patula, Asteraceae, was evaluated for its antifungal properties and analyzed by gas chromatography and gas chromatography–mass spectrometry. Thirty compounds were identified, representing 89.1% of the total detected. The main components were piperitone (24.74%), piperitenone (22.93%), terpinolene (7.8%), dihydro tagetone (4.91%), cis-tagetone (4.62%), limonene (4.52%), and allo-ocimene (3.66%). The oil exerted a good antifungal activity against two phytopathogenic fungi, Botrytis cinerea and Penicillium digitatum, providing complete growth inhibition at 10 μl/ml and 1.25 μl/ml, respectively. The contribution of the two main compounds, piperitone and piperitenone, to the antifungal efficacy was also evaluated and ultrastructural modifications in mycelia were observed via electron microscopy, evidencing large alterations in hyphal morphology and a multisite mechanism of action. Correspondence and reprints: Dipartimento delle Risorse Naturali e Culturali, Università di Ferrara, Corso Porta di Mare 2, 44100 Ferrara, Italy.     

Induction of antimicrobial 3-deoxyflavonoids in pome fruit trees controls fire blight

Fire blight, a devastating bacterial disease in pome fruits, causes severe economic losses worldwide. Hitherto, an effective control could only be achieved by using antibiotics, but this implies potential risks for human health, livestock and environment. A new approach allows transient inhibition of a step in the flavonoid pathway, thereby inducing the formation of a novel antimicrobial 3-deoxyflavonoid controlling fire blight in apple and pear leaves. This compound is closely related to natural phytoalexins in sorghum. The approach does not only provide a safe method to control fire blight: Resistance against different pathogens is also induced in other crop plants.