Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes

K_V10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of K_V10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of K_V10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length K_V10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of K_V10.1, which need to be considered when ion channels are targeted for cancer therapy.     

A conformational transition state accompanies tryptophan activation by B. stearothermophilus tryptophanyl-tRNA synthetase

B. stearothermophilus tryptophanyl-tRNA synthetase catalysis proceeds via high-energy protein conformations. Unliganded MD trajectories of the pretransition-state complex with Mg(2+)ATP and the (post) transition-state analog complex with adenosine tetraphosphate relax rapidly in opposite directions, the former regressing, the latter progressing along the structural reaction coordinate. The two crystal structures (rmsd 0.7 A) therefore lie on opposite sides of a conformational free-energy maximum as the chemical transition state forms. SNAPP analysis illustrates the complexity of the associated long-range conformational coupling. Switching interactions in four nonpolar core regions are locally isoenergetic throughout the transition. Different configurations, however, propagate their effects to unfavorable, longer-range interactions at the molecular surface. Designed mutation shows that switching interactions enhance the rate, perhaps by destabilizing the ground state immediately before the transition state and limiting nonproductive diffusion before and after the chemical transition state, thereby reducing the activation entropy. This paradigm may apply broadly to energy-transducing enzymes.     

Chromatographic profiles and antimicrobial activity of the essential oils obtained from some species and cultivars of the Mentheae tribe (Lamiaceae)

The present study was focused on the chemical composition and antimicrobial activity of the essential oils (EsO) obtained from five Lamiaceae representatives grown in the south of Ukraine. Among them are Salvia sclarea L., Monarda didyma (cultivar ‘Cambridge Scarlet’), Thymus pulegioides (cultivar ‘2/6-07’), Thymus vulgaris (cultivar ‘Jalos’), and Thymus serpyllum L. The component analysis of the EsO was carried out by gas chromatography method coupled with mass spectrometry (GC–MS). The antimicrobial properties of the EsO were determined using the agar diffusion test against widespread pathogenic bacterial strains (Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Streptococcus pyogenes) and opportunistic yeast Candida albicans. The EsO of Thymus serpyllum and Thymus vulgaris (cultivar ‘Jalos’) displayed noteworthy antibacterial properties against a wide spectrum of the microorganisms. These antimicrobial properties could be attributed to the high content of aromatic monoterpenoid thymol (52.56% and 47.33%, respectively). The EsO of Salvia sclarea with the dominance of linalyl acetate (45.51%) and linalool (38.98%) as well as Thymus pulegioides (cultivar ‘2/6-07’) containing α-citral (27.10%) and β-citral (17.11%) demonstrated the strongest antimicrobial effects on typical and clinical strains of Staphylococcus aureus with the inhibition zones in the range of 24.0–31.0 mm. The Salvia sclarea EsO demonstrated the most significant effect against clinical strains of Candida albicans. In conclusion, the present study revealed the chemical composition of five Lamiaceae species and cultivars grown in the south of Ukraine and considerable antimicrobial activity of the tested EsO, especially against the typical and clinical strains of Staphylococcus aureus and Candida albicans. The obtained results could be perspective for applying in the pharmaceutical industry and for the conservation of food and cosmetic products.     

Dark-Bright Exciton Splitting Dominates Low-Temperature Diffusion in Halide Perovskite Nanocrystal Assemblies

Semiconductor nanocrystals can replace conventional bulk materials completely in displays and light-emitting diodes. Exciton transport dominates over charge carrier transport for materials with high exciton binding energies and long ligands, such as halide perovskite nanocrystal films. Here, how beneficial superlattices – nearly perfect 3D assemblies of nanocrystals - are to exciton transport is investigated. Surprisingly, the high degree of order is not as crucial as the individual nanocrystal size, which strongly influences the splitting of the excitonic manifold into bright and dark states. At very low temperatures, the energetic splitting is larger for the smallest nanocrystals, and dark levels with low oscillator strength effectively trap excitons inside individual nanocrystals, suppressing diffusion. The effect is reversed at elevated temperatures, and the larger nanocrystal size becomes detrimental to exciton transport due to enhanced exciton trapping and dissociation. The results reveal that the nanocrystal size must be strongly accounted for in design strategies of future optoelectronic applications.     

Codon-optimized glucoamylase sGAI of Aspergillus awamori improves starch utilization in an industrial yeast

The development of a yeast that converts raw starch to ethanol in one step (called consolidated bioprocessing) could yield large cost reductions in the bioethanol industry. The aim of this study was to develop an efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production. A native and codon-optimized variant of the Aspergillus awamori glucoamylase gene were expressed in the S. cerevisiae Y294 laboratory strain. Codon optimization resulted to be effective and the synthetic sequence sGAI was then δ-integrated into a S. cerevisiae strain with promising industrial fermentative traits. The mitotically stable recombinant strains showed high enzymatic capabilities both on soluble and raw starch (2425 and 1140 nkat/g dry cell weight, respectively). On raw corn starch, the engineered yeasts exhibited improved fermentative performance with an ethanol yield of 0.42 (g/g), corresponding to 75?% of the theoretical maximum yield.     

Cross‐calibration of different radar systems for monitoring nocturnal bird migration across Europe and the Near East

Large parts of the continents are continuously scanned by terrestrial weather radars to monitor precipitation and wind conditions. These systems also monitor the mass movements of bird, bat, and insect migration, but it is still unknown how many of these systems perform with regard to detection and quantification of migration intensities of the different groups. In this study that was undertaken within five regions across Europe and the Middle East we examined to what extent bird migration intensities derived from different weather radars are comparable between each other and relate to intensities measured by local small‐scaled radars, some of them specifically developed to monitor birds. Good correspondence was found for the relative day‐to‐day pattern in migration intensities among most radar systems that were compared. Absolute intensities varied between different systems and regions. The findings of this study can be used to infer about absolute bird migration intensities measured by different radar systems and consequently help resolving methodological issues regarding the estimation of migrant numbers in the Western‐Palearctic region. It further depicts a scientific basis for the future monitoring of migratory bird populations across a large spatio‐temporal scale, predicting their movements and studying its consequences on ecological systems and human lives.     

DNA2 Cooperates with the WRN and BLM RecQ Helicases to Mediate Long-range DNA End Resection in Human Cells

The 5′-3′ resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5′-3′ DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.     

The Effect of Exogenous β-<Emphasis Type="Italic">N-</Emphasis>Methylamino-<Emphasis Type="SmallCaps">l</Emphasis>-alanine on the Growth of <Emphasis Type="Italic">Synechocystis</Emphasis> PCC6803

β-N-Methylamino-l-alanine (BMAA), a non-proteinogenic amino acid, has been detected in a range of cyanobacteria, including terrestrial, aquatic, free living and endosymbiotic species. The widespread occurrence of cyanobacteria in the environment raises concerns regarding the ecological and toxicological impact of BMAA, and consequently, studies have focussed extensively on the toxicity and environmental impact of BMAA, while no research has addressed the ecophysiological or metabolic role of the compound in cyanobacteria. In this study, both the uptake of exogenous BMAA by and the effect of exogenous BMAA on the growth of Synechocystis PCC6803 were investigated. BMAA was rapidly taken up by the non-diazotrophic cyanobacterium Synechocystis PCC6803 in a concentration dependent manner. The presence of exogenous BMAA resulted in a substantial and concentration-dependent decrease in cell growth and the substantial loss of photosynthetic pigmentation. Similar effects were seen in the presence of the non-proteinogenic amino acid, 2,4-diaminobutyric acid but to a lesser degree than that of BMAA. The effects were reversed when light was decreased from 16 to 10 μmol m⁻² s⁻¹. Control cultures grown in the presence of l-arginine, l-asparagine, l-glutamate and glycine showed normal or slightly increased growth with no change in pigmentation. The decrease in growth rate coupled to bleaching indicates that BMAA may induce chlorosis in the presence of adequate photosynthetic radiation suggesting a connection between BMAA and the induction of conditions, such as nitrogen or sulphur depletion, that result in growth arrest and the induction of chlorosis.     

Yeast adaptor protein, Nbp2p, is conserved regulator of fungal Ptc1p phosphatases and is involved in multiple signaling pathways

Nbp2p is an Src homology 3 (SH3) domain-containing yeast protein that is involved in a variety of cellular processes. This small adaptor protein binds to a number of different proteins through its SH3 domain, and a region N-terminal to the SH3 domain binds to the protein phosphatase, Ptc1p. Despite its involvement in a large number of physical and genetic interactions, the only well characterized function of Nbp2p is to recruit Ptc1p to the high osmolarity glycerol pathway, which results in down-regulation of this pathway. In this study, we have discovered that Nbp2p orthologues exist in all Ascomycete and Basidiomycete fungal genomes and that all possess an SH3 domain and a conserved novel Ptc1p binding motif. The ubiquitous occurrence of these two features, which we have shown are both critical for Nbp2p function in Saccharomyces cerevisiae, implies that a conserved role of Nbp2p in all of these fungal species is the targeting of Ptc1p to proteins recognized by the SH3 domain. We also show that in a manner analogous to its role in the high osmolarity glycerol pathway, Nbp2p functions in the down-regulation of the cell wall integrity pathway through SH3 domain-mediated interaction with Bck1p, a component kinase of this pathway. Based on functional studies on the Schizosaccharomyces pombe and Neurospora crassa Nbp2p orthologues and the high conservation of the Nbp2p binding site in Bck1p orthologues, this function of Nbp2p appears to be conserved across Ascomycetes. Our results also clearly imply a function for the Nbp2p-Ptc1p complex other cellular processes.     

α7 Nicotinic Acetylcholine Receptor-Specific Antibody Induces Inflammation and Amyloid β42 Accumulation in the Mouse Brain to Impair Memory

Nicotinic acetylcholine receptors (nAChRs) expressed in the brain are involved in regulating cognitive functions, as well as inflammatory reactions. Their density is decreased upon Alzheimer disease accompanied by accumulation of β-amyloid (Aβ₄₂), memory deficit and neuroinflammation. Previously we found that α7 nAChR-specific antibody induced pro-inflammatory interleukin-6 production in U373 glioblastoma cells and that such antibodies were present in the blood of humans. We raised a hypothesis that α7 nAChR-specific antibody can cause neuroinflammation when penetrating the brain. To test this, C57Bl/6 mice were either immunized with extracellular domain of α7 nAChR subunit α7(1-208) or injected with bacterial lipopolysaccharide (LPS) for 5 months. We studied their behavior and the presence of α3, α4, α7, β2 and β4 nAChR subunits, Aβ₄₀ and Aβ₄₂ and activated astrocytes in the brain by sandwich ELISA and confocal microscopy. It was found that either LPS injections or immunizations with α7(1-208) resulted in region-specific decrease of α7 and α4β2 and increase of α3β4 nAChRs, accumulation of Aβ₄₂ and activated astrocytes in the brain of mice and worsening of their episodic memory. Intravenously transferred α7 nAChR-specific-antibodies penetrated the brain parenchyma of mice pre-injected with LPS. Our data demonstrate that (1) neuroinflammation is sufficient to provoke the decrease of α7 and α4β2 nAChRs, Aβ₄₂ accumulation and memory impairment in mice and (2) α7(1-208) nAChR-specific antibodies can cause inflammation within the brain resulting in the symptoms typical for Alzheimer disease.