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.     

Do well-connected landscapes promote road-related mortality?

Cost surface (CS) models have emerged as a useful tool to examine the interactions between landscapes patterns and wildlife at large-scale extents. This approach is particularly relevant to guide conservation planning for species that show vulnerability to road networks in human-dominated landscapes. In this study, we measured the functional connectivity of the landscape in southern Portugal and examined how it may be related to stone marten road mortality risk. We addressed three questions: (1) How different levels of landscape connectivity influence stone marten occurrence in montado patches? (2) Is there any relation between montado patches connectivity and stone marten road mortality risk? (3) If so, which road-related features might be responsible for the species’ high road mortality? We developed a series of connectivity models using CS scenarios with different resistance values given to each vegetation cover type to reflect different resistance to species movement. Our models showed that the likelihood of occurrence of stone marten decreased with distance to source areas, meaning continuous montado. Open areas and riparian areas within open area matrices entailed increased costs. We found higher stone marten mortality on roads in well-connected areas. Road sinuosity was an important factor influencing the mortality in those areas. This result challenges the way that connectivity and its relation to mortality has been generally regarded. Clearly, landscape connectivity and road-related mortality are not independent.     

Essential oil production in shoot cultures versus field-grown plants of Thymus caespititius

Thymus caespititius Brot. is an important aromatic species, due to synthesis and production of essential oils for the pharmaceutical and food industries. In the present study, levels of essential oils from two chemotypes, including carvacrol/thymol (CT) and sabinene/carvacrol (SC), were evaluated in proliferating shoot cultures (6–12 subcultures following establishment) and compared to those from field-grown plants. The essential oils were isolated by hydrodistillation and analysed by gas chromatography (GC) and GC–mass spectrometry (GC–MS). Cultures grown under in vitro culture conditions, evaluated over six subcultures, were found to maintain stable composition of essential oils. For the CT chemotype, carvacrol (42 %) and thymol (23 %) were the main essential oil components detected in field-grown plants; in proliferating shoot cultures the levels detected attained 17–25 % in the case of carvacrol and 18–23 % in that of thymol, closely followed by carvacryl acetate (15–23 %) and thymyl acetate (11–15 %). For the SC chemotype, carvacrol (13–28 %), sabinene (18–45 %), and thymol (9–12 %) were the main essential oil components detected in both field-grown and proliferating shoot cultures. Our experiments showed that the essential oil composition in proliferating shoot cultures was not only stable, but also qualitatively similar to that of field-grown plants, notwithstanding minor quantitative differences.     

23Na multiple quantum filtered NMR characterisation of Na+ binding and dynamics in animal cells: a comparative study and effect of Na+/Li+ competition

Double quantum and triple quantum filtered ²³Na nuclear magnetic resonance techniques were used to characterise in detail the isotropic and anisotropic binding and dynamics of intra- and extracellular Na⁺ in different cellular systems, in the absence and presence of Li⁺. The kinetics of Li⁺ influx by different cell types was evaluated. At steady state, astrocytes accumulated more Li⁺ than red blood cells (RBCs), while a higher intracellular Li⁺ concentration was found in chromaffin than in SH-SY5Y cells. Anisotropic and isotropic motions were detected for extracellular Na⁺ in all cellular systems studied. Isotropic intracellular Na⁺ motions were observed in all types of cells, while anisotropic Na⁺ motions in the intracellular compartment were only detected in RBCs. ²³Na triple quantum signal efficiency for intracellular Na⁺ was SH-SY5Y > chromaffin > RBCs, while the reverse order was observed for the extracellular ions. ²³Na double quantum signal efficiency for intracellular Na⁺ was non-zero only in RBCs, and for extracellular Na⁺ the order RBCs > chromaffin > SH-SY5Y cells was observed. Li⁺ loading generally decreased intracellular Na⁺ isotropic movements in the cells, except for astrocytes incubated with a low Li⁺ concentration and increased anisotropic intracellular Na⁺ movements in RBCs. Li⁺ effects on the extracellular signals were more complex, reflecting Li⁺/Na⁺ competition for isotropic and anisotropic binding sites at the extracellular surface of cell membranes and also at the surface of the gel used for cell immobilisation. These results are relevant and contribute to the interpretation of the in vivo pharmacokinetics and sites of Li⁺ action.     

Excitotoxic stimulation downregulates the ubiquitin–proteasome system through activation of NMDA receptors in cultured hippocampal neurons

Overactivation of glutamate receptors contributes to neuronal damage (excitotoxicity) in ischemic stroke but the detailed mechanisms are not fully elucidated. Brain ischemia is also characterized by an impairment of the activity of the proteasome, one of the major proteolytic systems in neurons. We found that excitotoxic stimulation with glutamate rapidly decreases ATP levels and the proteasome activity, and induces the disassembly of the 26S proteasome in cultured rat hippocampal neurons. Downregulation of the proteasome activity, leading to an accumulation of ubiquitinated proteins, was mediated by calcium entry through NMDA receptors and was only observed in the nuclear fraction. Furthermore, excitotoxicity-induced proteasome inhibition was partially sensitive to cathepsin-L inhibition and was specifically induced by activation of extrasynaptic NMDA receptors. Oxygen and glucose deprivation induced neuronal death and downregulated the activity of the proteasome by a mechanism dependent on the activation of NMDA receptors. Since deubiquitinating enzymes may regulate proteins half-life by counteracting ubiquitination, we also analyzed how their activity is regulated under excitotoxic conditions. Glutamate stimulation decreased the total deubiquitinase activity in hippocampal neurons, but was without effect on the activity of Uch-L1, showing that not all deubiquitinases are affected. These results indicate that excitotoxic stimulation with glutamate has multiple effects on the ubiquitin–proteasome system which may contribute to the demise process in brain ischemia and in other neurological disorders.     

Molecular and cytological characterization of genomic variability in hexaploid wheat 'Lindstr?m'.

'Lindstr?m' wheat (AABBDD+rye B chromosomes) was used to study the effects of alien chromatin introgressed into a wheat genetic background, subjecting the wheat genome to a new and transient allopolyploidisation episode. Using this experimental material, we have previously demonstrated that no large-scale chromosomal translocations occurred as a result of the genomic constitution of the addition line. However, we have shown that the presence of a number of rye B chromosomes is associated with changes in the interphase organization and expression patterns of wheat rDNA loci. We have now extended our studies to focus on a further characterization of 'Lindstr?m' 5S rDNA loci and also on high molecular weight glutenin subunit (HMW-GS) patterns. In the process, we have uncovered an unusually large variant of the 5S rDNA locus on wheat chromosome 1B (not to be confused with rye B chromosomes) and 2 novel HMW glutenin y-type alleles. These changes are not directly related to variation in rye B chromosome number in the present material, but the fact that a new, and still segregating, 1Dy HMW-GS gene was identified indicates a recent timescale for its origin. Strikingly, the 'Lindstr?m' 5S rDNA 1B locus integrates a unit sharing 94% homology with a rye 5S rDNA sequence, suggesting the possibility that the wheat locus was colonized by highly homologous rye sequences during the breeding of 'Lindstr?m', when the rye and wheat genomes were together, albeit briefly, in the same nucleus.     

The conserved sequence NXX[S/T]HX[S/T]QDXXXT of the lactate/pyruvate:H(+) symporter subfamily defines the function of the substrate translocation pathway

In Saccharomyces cerevisiae Jen1p is a lactate/proton symporter belonging to the lactate/pyruvate:H(+) symporter subfamily (TC#2.A.1.12.2) of the Major Facilitator Superfamily. We investigated structure-function relationships of Jen1p using a rational mutational analysis based on the identification of conserved amino acid residues. In particular, we studied the conserved sequence (379)NXX[S/T]HX[S/T]QDXXXT(391). Substitution of amino acid residues N379, H383 or D387, even with very similar amino acids, resulted in a dramatic reduction of lactate and pyruvate uptake, but conserved measurable acetate transport. Acetate transport inhibition assays showed that these mutants conserve the ability to bind, but do not transport, lactate and pyruvate. More interestingly, the double mutation H383D/D387H, while behaving as a total loss-of-function allele for lactate and pyruvate uptake, can fully restore the kinetic parameters of Jen1p for acetate transport. Thus, residues N379, H383 or D387 affect both the transport capacity and the specificity of Jen1p. Substitutions of Q386 and T391 resulted in no or moderate changes in Jen1p transport capacities for lactate, pyruvate and acetate. On the other hand, Q386N reduces the binding affinities for all Jen1p substrates, while Q386A increases the affinity specifically for pyruvate. We also tested Jen1p specificity for a range of monocarboxylates. Several of the mutants studied showed altered inhibition constants for these acids. These results and 3D in silico modelling by homology threading suggest that the conserved motif analyzed is part of the substrate translocation pathway in the lactate/pyruvate:H(+) symporter subfamily.