A screen for cold-induced plant metabolites that promote RNA folding

Die Anpassung von Pflanzen an Kältestress beinhaltet eine drastische Änderung des Metabolit- und Proteinprofils. Neben anderen Proteinen werden auch RNA-Chaperone vermehrt exprimiert. RNA-Chaperone können RNA-RNA Interaktionen lösen und dadurch der Zelle helfen, die erhöhte thermodynamische Stabilität von RNA Strukturen bei niedriger Temperatur zu kompensieren. Dagegen ist über die Funktion von Metaboliten während der Kälteadaption wenig bekannt. Eine gut untersuchte Gruppe stress-induzierter Metaboliten sind Osmolyte. Osmolyte halten die Osmolarität der Zelle aufrecht, ohne jedoch mit Makromolekülen der Zelle zu interagieren. In hohen Konzentrationen verändern jedoch viele kälte-induzierte Metaboliten die Stabilität von Proteinen und Nukleinsäuren. In dieser Studie wurde getestet, ob diese Metaboliten als RNA-Chaperone fungieren können, indem sie den Austausch zwischen RNA-Strängen beschleunigen. Weder individuelle kälte-induzierte Metaboliten noch Metabolitgruppen beschleunigen die Hybridisierung von oder den Austausch zwischen kurzen RNA Oligonukleotiden. Es konnte ebenfalls kein Einfluss auf die strukturelle Stabilität von RNA festgestellt werden. Um eine physiologische Metabolitzusammensetzung zu approximieren, wurden Metabolitextrakte von kältebehandelten und nicht-kältebahandelten Chlamydomonas reinhardtii Zellen hergestellt und verglichen. Beide Extrakte fördern die korrekte Faltung von RNA im selben Ausmaß und beschleunigen den Austausch zwischen kurzen RNA Oligonukeotiden. Allerdings ist diese Beschleunigung in Kälteschockextrakten, im Vergleich zu Kontrollextrakten schon bei niedrigeren Metabolitkonzentrationen zu beobachten. Ein Protokoll für die Aufreinigung von GRP7, einem kälte-induzierten Protein welches vermutlich RNA-Chaperon Aktivität aufweist, wurde entwickelt. Zusammenfassend konnte kein Einfluss von individuellen kälte-induzierten Metaboliten auf die RNA-Faltung festgestellt werden. Kälte-Schock Extrakte beschleunigen den Austausch zwischen RNA Strängen in vitro. Dies weist darauf hin, dass die Änderung des Metabolitprofils während Kälteschock einen Einfluss auf RNA haben könnte. Allerdings lassen diese Ergebnisse keine Schlussfolgerung zu, ob dieser Einfluss von biologischer Relevanz ist.Low temperature is a major stress factor for plants. The plants response to this kind of stress involves a dramatic change in the metabolite and protein profile. Among other cold-inducible proteins, RNA chaperones are highly expressed. RNA chaperones are able to resolve RNA-RNA interactions and thereby help the cell to cope with the increased thermodynamic stability of non-native RNA structures at low temperature. The change in the metabolite profile is poorly understood. One major group of upregulated metabolites are “osmolytes”, a group of metabolites that maintain the cells osmolarity but do not show any interaction with macromolecules. However, many cold-inducible metabolites were shown to influence the thermodynamic stability of proteins and nucleic acids. This study tested whether these metabolites can help the plant cell during cold stress by acting as RNA chaperones. Individual cold-shock metabolites as well as groups of cold-shock metabolites do not fulfill the requirements to enhance annealing or strand-displacement of short RNA oligonucleotides in a FRET-based assay. Approximately physiological concentrations of these metabolites also did not alter RNA stability nor the shape of the melting transition. In order to approximate physiological metabolite combinations, polar metabolites were extracted from cold-treated Chlamydomonas reinhardtii cells. The extracts were tested for their ability to promote the functional conformation of a folding retarded group I intron in a trans-splicing assay. The splicing was equally efficient in extracts from cold-treated cells and from not-cold-treated cells. Both extracts were found to enhance the strand-displacement reaction between a 21mer duplex RNA and a 32mer fully complementary competitor. The cold-shock extract enhanced the displacement-reaction already at lower concentrations. In addition, a protocol for a tag-free purification of the proposed RNA chaperone GRP7 was developed. In conclusion, cold-shock metabolites were not found to influence RNA folding. However, the change in the metabolite profile upon cold-shock can have an influence on RNA in vitro as shown in the strand-displacement assay. Whether this influence is of biological relevance cannot be concluded

Tubulins and brain development: The origins of functional specification

The development of the vertebrate central nervous system is reliant on a complex cascade of biological processes that include mitotic division, relocation of migrating neurons, and the extension of dendritic and axonal processes. Each of these cellular events requires the diverse functional repertoire of the microtubule cytoskeleton for the generation of forces, assembly of macromolecular complexes and transport of molecules and organelles. The tubulins are a multi-gene family that encode for the constituents of microtubules, and have been implicated in a spectrum of neurological disorders. Evidence is building that different tubulins tune the functional properties of the microtubule cytoskeleton dependent on the cell type, developmental profile and subcellular localisation. Here we review of the origins of the functional specification of the tubulin gene family in the developing brain at a transcriptional, translational, and post-transcriptional level. We remind the reader that tubulins are not just loading controls for your average Western blot

Measurement of the Free-Energy Dependence of Interfacial Charge-Transfer Rate Constants using ZnO/H_2O Semiconductor/Liquid Contacts

The dependence of electron-transfer rate constants on the driving force for interfacial charge transfer has been investigated using n-type ZnO electrodes in aqueous solutions. Differential capacitance versus potential and current density versus potential measurements were used to determine the energetics and kinetics, respectively, of the interfacial electron-transfer processes. A series of nonadsorbing, one-electron, outer-sphere redox couples with formal reduction potentials that spanned approximately 900 mV allowed evaluation of both the normal and Marcus inverted regions of interfacial electron-transfer processes. All rate processes were observed to be kinetically first-order in the concentration of surface electrons and first-order in the concentration of dissolved redox acceptors. The band-edge positions of the ZnO were essentially independent of the Nernstian potential of the solution over the range 0.106−1.001 V vs SCE. The rate constant at optimal exoergicity was observed to be approximately 10^(-16) cm4 s^(-1). The rate constant versus driving force dependence at n-type ZnO electrodes exhibited both normal and inverted regions, and the data were well-fit by a parabola generated using classical Marcus theory with a reorganization energy of 0.67 eV. NMR line broadening measurements of the self-exchange rate constants indicated that the redox couples had reorganization energies of 0.64−0.69 eV. The agreement between the reorganization energy of the ions in solution and the reorganization energy for the interfacial electron-transfer processes indicated that the reorganization energy was dominated by the redox species in the electrolyte, as expected from an application of Marcus theory to semiconductor electrodes

The RNA annealing mechanism of the HIV-1 Tat peptide: conversion of the RNA into an annealing-competent conformation

The annealing of nucleic acids to (partly) complementary RNA or DNA strands is involved in important cellular processes. A variety of proteins have been shown to accelerate RNA/RNA annealing but their mode of action is still mainly uncertain. In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44–61), which accelerates the reaction efficiently. The activity of the peptide is strongly regulated by mono- and divalent cations which hints at the importance of electrostatic interactions between RNA and peptide. Mutagenesis of the peptide illustrated the dominant role of positively charged amino acids in RNA annealing—both the overall charge of the molecule and a precise distribution of basic amino acids within the peptide are important. Additionally, we found that Tat(44–61) drives the RNA annealing reaction via entropic rather than enthalpic terms. One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal

The European wood pellets market

EU set an ambitious program on renewable energies until 2020. Biomass will play an important role. Within the biomass sector, wood pellets are an upcoming biofuel market. Recently wood pellets are commonly made of by-products of sawmill and wood processing industry, especially for small scale heating appliances. The availability of sawmill by-products depends on the demand of sawnwood products. Sawmill is the bottleneck in the pellets logistics chain. The question is, in what extent raw material supply is able to meet the rising demand on wood pellets and what are possible alternatives to "classical" wood pellets. Therefore an assessment of European forestry and sawnwood industry on the basis of several surveys was made. The results are compared with an estimation of pellets demand according to the biomass targets of EU policies. An analysis of total costs for pellets production shows the influence of raw material costs. The expected demand for wood pellets for medium scale heating, derived from the objectives of the EU climate and energy package, will not be covered by the standards of corresponding premium wood pellets. Even in the minimum scenario (10% of biomass heat demand is covered by the use of pellets and here again about 50% of that exclusively in small scale heating), 14 million tons of pellets will be required. The pellets supply that was derived from the sawmill industry forecast amounts to 8 million tons. The raw material supply has to be widened. Pellets have to be produced from forestry residuals, from short rotation plantation on agricultural farmland and from agricultural products like grain, different types of grass, agricultural residuals etc. New standard for non-wood pellets has to be developed to create a transparent and efficient market for different types of pellets for different purposes. Boiler technology has to be improved for alternative pellets types to meet the regulations of flue gas emissions and to meet the technical requirements for more aggressive components.8

Measurement of the Dependence of Interfacial Charge-Transfer Rate Constants on the Reorganization Energy of Redox Species at n-ZnO/H_2O Interfaces

The interfacial energetic and kinetics behavior of n-ZnO/H_2O contacts have been determined for a series of compounds, cobalt trisbipyridine (Co(bpy)_3^(3+/2+)), ruthenium pentaamine pyridine (Ru(NH_3)_5py^(3+/2+)), cobalt bis-1,4,7-trithiacyclononane (Co(TTCN)_2^(3+/2+)), and osmium bis-dimethyl bipyridine bis-imidazole (Os(Me_2bpy)_2(Im)_2^(3+/2+)), which have similar formal reduction potentials yet which have reorganization energies that span approximately 1 eV. Differential capacitance vs potential and current density vs potential measurements were used to measure the interfacial electron-transfer rate constants for this series of one-electron outer-sphere redox couples. Each interface displayed a first-order dependence on the concentration of redox acceptor species and a first-order dependence on the concentration of electrons in the conduction band at the semiconductor surface, in accord with expectations for the ideal model of a semiconductor/liquid contact. Rate constants varied from 1 × 10^(-19) to 6 × 10^(-17) cm^4 s^(-1). The interfacial electron-transfer rate constant decreased as the reorganization energy, λ, of the acceptor species increased, and a plot of the logarithm of the electron-transfer rate constant vs (λ + ΔG°‘)^2/4λk_BT (where ΔG°‘ is the driving force for interfacial charge transfer) was linear with a slope of ∼ −1. The rate constant at optimal exoergicity was found to be ∼5 × 10^(-17) cm^4 s^(-1) for this system. These results show that interfacial electron-transfer rate constants at semiconductor electrodes are in good agreement with the predictions of a Marcus-type model of interfacial electron-transfer reactions