Pimecrolimus Is a Potent Inhibitor of Allergic Reactions to Hymenopteran Venom Extracts and Birch Pollen Allergen In Vitro

Pimecrolimus (Elidel, SDZ ASM 981) is an anti-inflammatory and immunomodulatory 33-epichloro-derivative of macrolactam ascomycin, with low potential for affecting systemic immune responses compared with other calcineurin inhibitors, cyclosporin A and tacrolimus. Despite numerous studies focused on the mechanism of pimecrolimus action on mast cells, only the single report has addressed pimecrolimus effects on other typical FcεRI-expressing cells, the basophils. Patients allergic to birch pollen (n = 20), hymenopteran venoms (n = 23) and 10 non-allergic volunteers were examined. Primary human basophils pre-treated or not with 0.5–50 μMol pimecrolimus were exposed to various concentrations of recombinant Bet v 1a allergen, bee or wasp venom extracts and anti-IgE for 20 min, and then examined for the expression of CD45, CD193, CD203c, CD63 and CD164 using flow cytometry. The externalization of basophil activation markers (CD63 and CD164) was equally inhibited through pimecrolimus in cells activated by recombinant pollen allergen, hymenopteran venom extracts and anti-IgE. Although the individual response rate was subject to strong variation, importantly, pre-treatment with pimecrolimus lowered the number of activated basophils in response to any of the stimuli in the basophils from all patients. The inhibition was concentration-dependent; approximately half of the basophils were inhibited in the presence of 2.5 mMol pimecrolimus. Pimecrolimus is a valuable new tool for the inhibition of hyper-reactive basophils in patients with pollen allergy and a history of anaphylactic reactions to bee or wasp venoms. Further research should address short-term use of pimecrolimus in vivo in a wide spectrum of allergic diseases.     

Characterization of Adhesive Exopolysaccharide (EPS) Produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Under Starvation Conditions

Pseudomonas aeruginosa synthesizes large quantities of exopolysaccharide (EPS), making it an excellent model organism for the study of EPS-mediated adhesion. The purpose of this investigation was to evaluate the influence of limited nutrients availability in the culture medium on the composition of EPS produced by P. aeruginosa. The relationship between the EPS production and the adhesion process of the P. aeruginosa cells to stainless steel surface (type 316 L) under starvation conditions were also examined. In all experimental variants P. aeruginosa produced more EPS with an increase of incubation period upon starvation conditions. Under limited nutrients condition, glucose dominated in the EPS materials. After 6 days of the process, only glucosyl units were detected in the extracellular matrix produced by nutrient-deprived P. aeruginosa cells. These extracellular molecules promoted more advanced stages of P. aeruginosa biofilm formation on the surface of stainless steel.     

BAG1 restores formation of functional DJ-1 L166P dimers and DJ-1 chaperone activity

Mutations in the gene coding for DJ-1 protein lead to early-onset recessive forms of Parkinson’s disease. It is believed that loss of DJ-1 function is causative for disease, although the function of DJ-1 still remains a matter of controversy. We show that DJ-1 is localized in the cytosol and is associated with membranes and organelles in the form of homodimers. The disease-related mutation L166P shifts its subcellular distribution to the nucleus and decreases its ability to dimerize, impairing cell survival. Using an intracellular foldase biosensor, we found that wild-type DJ-1 possesses chaperone activity, which is abolished by the L166P mutation. We observed that this aberrant phenotype can be reversed by the expression of the cochaperone BAG1 (Bcl-2–associated athanogene 1), restoring DJ-1 subcellular distribution, dimer formation, and chaperone activity and ameliorating cell survival.     

Human RioK3 is a novel component of cytoplasmic pre-40S pre-ribosomal particles

Maturation of the 40S ribosomal subunit precursors in mammals mobilizes several non-ribosomal proteins, including the atypical protein kinase RioK2. Here, we have investigated the involvement of another member of the RIO kinase family, RioK3, in human ribosome biogenesis. RioK3 is a cytoplasmic protein that does not seem to shuttle between nucleus and cytoplasm via a Crm1-dependent mechanism as does RioK2 and which sediments with cytoplasmic 40S ribosomal particles in a sucrose gradient. When the small ribosomal subunit biogenesis is impaired by depletion of either rpS15, rpS19 or RioK2, a concomitant decrease in the amount of RioK3 is observed. Surprisingly, we observed a dramatic and specific increase in the levels of RioK3 when the biogenesis of the large ribosomal subunit is impaired. A fraction of RioK3 is associated with the non ribosomal pre-40S particle components hLtv1 and hEnp1 as well as with the 18S-E pre-rRNA indicating that it belongs to a bona fide cytoplasmic pre-40S particle. Finally, RioK3 depletion leads to an increase in the levels of the 21S rRNA precursor in the 18S rRNA production pathway. Altogether, our results strongly suggest that RioK3 is a novel cytoplasmic component of pre-40S pre-ribosomal particle(s) in human cells, required for normal processing of the 21S pre-rRNA.     

Spatial coordination of kindlin-2 with talin head domain in interaction with integrin β cytoplasmic tails

Both talin head domain and kindlin-2 interact with integrin β cytoplasmic tails, and they function in concert to induce integrin activation. Binding of talin head domain to β cytoplasmic tails has been characterized extensively, but information on the interaction of kindin-2 with this integrin segment is limited. In this study, we systematically examine the interactions of kindlin-2 with integrin β tails. Kindlin-2 interacted well with β(1) and β(3) tails but poorly with the β(2) cytoplasmic tail. This binding selectivity was determined by the non-conserved residues, primarily the three amino acids at the extreme C terminus of the β(3) tail, and the sequence in β(2) was non-permissive. The region at the C termini of integrin β(1) and β(3) tails recognized by kindlin-2 was a binding core of 12 amino acids. Kindlin-2 and talin head do not interact with one another but can bind simultaneously to the integrin β(3) tail without enhancing or inhibiting the interaction of the other binding partner. Kindlin-2 itself failed to directly unclasp integrin α/β tail complex, indicating that kindlin-2 must cooperate with talin to support the integrin activation mechanism.     

Minisatellite telomeres occur in the family Alliaceae but are lost in Allium

Although telomere sequences are considered to be highly conserved, there are switch-points in plant telomere evolution that are congruent with species' phylogenies. When Asparagales diverged, the Arabidopsis-type telomeric minisatellite repeat (TTTAGGG)(n) was first replaced by a human-type (TTAGGG)(n) repeat, and both were lost in Allium cepa (Alliaceae). We aimed to discover (1) when this loss occurred during divergence of Alliaceae and, (2) if (TTAGGG)(n) repeats were replaced by other known telomeric minisatellites. Slot-blot hybridization, fluorescent in situ hybridization, BAL31 digestion, asymmetric PCR, and cloning were used to identify and localize candidate telomeric sequences in species of Nothoscordum, Miersia, Ipheion, Tulbaghia, Gethyum, Gilliesia, Leucocoryne, Tristagma, and representatives of the three major Allium clades. Alliaceae genera other than Allium have human (TTAGGG)-type telomeric repeats that form telomeres. In Allium, only Tetrahymena-type (TTGGGG) repeats were ubiquitous in the genome, but they were not localized to telomeres. Likewise, the consensus telomeric repeats in Arabidopsis, human, Bombyx (TTAGG), Chlamydomonas (TTTTAGGG), and Oxytricha (TTTTGGGG) are absent in Allium telomeres. Alliaceae with human-type telomeres share telomere structures with related Asparagales species. We demonstrate that in the Allium ancestor human-type telomeric repeats were lost from telomeres and were not replaced by any investigated alternative minisatellite repeats. However, human and other types of minisatellite telomeric repeats are interspersed in some Allium genomes and their genomic signatures coincide with Allium clades.     

Tyrosine Phosphorylation of Integrin β3 Regulates Kindlin-2 Binding and Integrin Activation

Kindlins are essential for integrin activation in cell systems and do so by working in a cooperative fashion with talin via their direct interaction with integrin β cytoplasmic tails (CTs). Kindlins interact with the membrane-distal NxxY motif, which is distinct from the talin-binding site within the membrane-proximal NxxY motif. The Tyr residues in both motifs can be phosphorylated, and it has been suggested that this modification of the membrane-proximal NxxY motif negatively regulates interaction with the talin head domain. However, the influence of Tyr phosphorylation of the membrane-distal NxxY motif on kindlin binding is unknown. Using mutational analyses and phosphorylated peptides, we show that phosphorylation of the membrane-distal NITY⁷⁵⁹ motif in the β₃ CT disrupts kindlin-2 recognition. Phosphorylation of this membrane-distal Tyr also disables the ability of kindlin-2 to coactivate the integrin. In direct binding studies, peptides corresponding to the non-phosphorylated β₃ CT interacted well with kindlin-2, whereas the Tyr⁷⁵⁹-phosphorylated peptide failed to bind kindlin-2 with measurable affinity. These observations indicate that transitions between the phosphorylated and non-phosphorylated states of the integrin β₃ CT determine reactivity with kindlin-2 and govern the role of kindlin-2 in regulating integrin activation.     

Bacterial Endophytes from Vitis vinifera L. – Metabolomics Characterization of Plant-Endophyte Crosstalk

Bacterial endophytes are known to protect Vitis vinifera L. against various harmful effects of the environment and support its growth. However, for the most part, biochemical responses of such co-existence have not yet been fully elucidated. In this work, we aimed to characterize the activities of endophytic consortia in a plant-endophyte extract by measuring five indicators of colonization (overall endophyte metabolic activity, microbial ACC deaminase activity, ability to solubilize phosphorus, ability to convert atmospheric nitrogen to ammonia ions, and ability to produce growth promoting indole acetic acid), and find relationships between these activities and metabolome. The V. vinifera canes for the metabolomics fingerprinting were extracted successively with water and methanol, and analysed by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry. For data processing, the MS-DIAL – MS-CleanR – MS-FINDER software platform was used, and the data matrix was processed by PCA and PLS-DA multivariate statistical methods. The metabolites that were upregulated with the heavy endophyte colonization were mainly chlorins, phenolics, flavonoid and terpenoid glycosides, tannins, dihydropyranones, sesquiterpene lactones, and long-chain unsaturated fatty acids.     

Effect of cold and menthol on membrane potential in plants

'The Holy Grail' of plant ecology is to uncover rules that associate species and traits with environmental constraints, community composition and subsequent ecosystem functioning. These aims have been crystallized in recent years within the context of global climate change and environmental pollution, increasing the urgency of the need to predict how vegetation will respond across spatial scales. We investigated whether genetic diversity is associated with the way in which phenotypic plasticity within plant populations is realized and whether this is related to genotype abundance. We used environmental metabolomics to demonstrate biochemical variation between co-occurring genotypes of Carex caryophyllea L. A novel combined metabolomic/functional trait analysis was used to test the functionality of this variation in governing plasticity to variation in edaphic conditions, with particular reference to metabolic pathways that play important roles in growth-related traits. We show that genetic diversity within a wild C. caryophyllea population relates to differences in metabolic composition and functional traits in response to soil nutrient variation, influencing genotype abundance within a community. Our findings highlight the vital role genetic diversity plays within a population in facilitating plant phenotypic plasticity and the potential usefulness of environmental metabolomics to future ecological studies.