Binding to G-quadruplex RNA activates the mitochondrial GTPase NOA1

NOA1 is an evolutionary conserved, nuclear encoded GTPase essential for mitochondrial function and cellular survival. The function of NOA1 for assembly of mitochondrial ribosomes and regulation of OXPHOS activity depends on its GTPase activity, but so far no ligands have been identified that regulate the GTPase activity of NOA1. To identify nucleic acids that bind to the RNA-binding domain of NOA1 we employed SELEX (Systemic Evolution of Ligands by EXponential Enrichment) using recombinant mouse wildtype NOA1 and the GTPase mutant NOA1-K353R. We found that NOA1 binds specifically to oligonucleotides that fold into guanine tetrads (G-quadruplexes). Binding of G-quadruplex oligonucleotides stimulated the GTPase activity of NOA1 suggesting a regulatory link between G-quadruplex containing RNAs, NOA1 function and assembly of mitochondrial ribosomes.     

Rapid Assessment of Tablet Film Coating Quality by Multispectral UV Imaging

Chemical imaging techniques are beneficial for control of tablet coating layer quality as they provide spectral and spatial information and allow characterization of various types of coating defects. The purpose of this study was to assess the applicability of multispectral UV imaging for assessment of the coating layer quality of tablets. UV images were used to detect, characterize, and localize coating layer defects such as chipped parts, inhomogeneities, and cracks, as well as to evaluate the coating surface texture. Acetylsalicylic acid tablets were prepared on a rotary tablet press and coated with a polyvinyl alcohol-polyethylene glycol graft copolymer using a pan coater. It was demonstrated that the coating intactness can be assessed accurately and fast by UV imaging. The different types of coating defects could be differentiated and localized based on multivariate image analysis and Soft Independent Modeling by Class Analogy applied to the UV images. Tablets with inhomogeneous texture of the coating could be identified and distinguished from those with a homogeneous surface texture. Consequently, UV imaging was shown to be well-suited for monitoring of the tablet coating layer quality. UV imaging is a promising technique for fast quality control of the tablet coating because of the high data acquisition speed and its nondestructive analytical nature.     

Assessing the utility of conserving evolutionary history

It is often claimed that conserving evolutionary history is more efficient than species‐based approaches for capturing the attributes of biodiversity that benefit people. This claim underpins academic analyses and recommendations about the distribution and prioritization of species and areas for conservation, but evolutionary history is rarely considered in practical conservation activities. One impediment to implementation is that arguments related to the human‐centric benefits of evolutionary history are often vague and the underlying mechanisms poorly explored. Herein we identify the arguments linking the prioritization of evolutionary history with benefits to people, and for each we explicate the purported mechanism, and evaluate its theoretical and empirical support. We find that, even after 25 years of academic research, the strength of evidence linking evolutionary history to human benefits is still fragile. Most – but not all – arguments rely on the assumption that evolutionary history is a useful surrogate for phenotypic diversity. This surrogacy relationship in turn underlies additional arguments, particularly that, by capturing more phenotypic diversity, evolutionary history will preserve greater ecosystem functioning, capture more of the natural variety that humans prefer, and allow the maintenance of future benefits to humans. A surrogate relationship between evolutionary history and phenotypic diversity appears reasonable given theoretical and empirical results, but the strength of this relationship varies greatly. To the extent that evolutionary history captures unmeasured phenotypic diversity, maximizing the representation of evolutionary history should capture variation in species characteristics that are otherwise unknown, supporting some of the existing arguments. However, there is great variation in the strength and availability of evidence for benefits associated with protecting phenotypic diversity. There are many studies finding positive biodiversity–ecosystem functioning relationships, but little work exists on the maintenance of future benefits or the degree to which humans prefer sets of species with high phenotypic diversity or evolutionary history. Although several arguments link the protection of evolutionary history directly with the reduction of extinction rates, and with the production of relatively greater future biodiversity via increased adaptation or diversification, there are few direct tests. Several of these putative benefits have mismatches between the relevant spatial scales for conservation actions and the spatial scales at which benefits to humans are realized. It will be important for future work to fill in some of these gaps through direct tests of the arguments we define here.     

Biased random walk by stochastic fluctuations of chemoattractant-receptor interactions at the lower limit of detection

Binding of ligand to its receptor is a stochastic process that exhibits fluctuations in time and space. In chemotaxis, this leads to a noisy input signal. Therefore, in a gradient of chemoattractant, the cell may occasionally experience a "wrong" gradient of occupied receptors. We obtained a simple equation for P(pos), the probability that half of the cell closest to the source of chemoattractant has higher receptor occupancy than the opposite half of the cell. P(pos) depends on four factors, the gradient property delC/sq. root of C, the receptor characteristic R(t)/K(D), a time-averaging constant I, and nonreceptor noise sigma(B). We measured chemotaxis of Dictyostelium cells to known shallow gradients of cAMP and obtained direct estimates for these constants. Furthermore, we observed that in shallow gradients, the measured chemotaxis index is correlated with P(pos), which suggests that chemotaxis in shallow gradients is a pure biased random walk. From the observed chemotaxis and derived time-averaging constant, we deduce that the gradient transducing second messenger has a lifetime of 2-8 s and a diffusion rate constant of approximately 1 microm(2)/s. Potential candidates for such second messengers are discussed.     

Selection of CD8+ T cells with highly focused specificity during viral persistence in the central nervous system

The relationships between T cell populations during primary viral infection and persistence are poorly understood. Mice infected with the neurotropic JHMV strain of mouse hepatitis virus mount potent regional CTL responses that effectively reduce infectious virus; nevertheless, viral RNA persists in the central nervous system (CNS). To evaluate whether persistence influences Ag-specific CD8+ T cells, functional TCR diversity was studied in spleen and CNS-derived CTL populations based on differential recognition of variant peptides for the dominant nucleocapsid epitope. Increased specificity of peripheral CTL from persistently infected mice for the index epitope compared with immunized mice suggested T cell selection during persistence. This was confirmed with CD8+ T cell clones derived from the CNS of either acutely (CTLac) or persistently (CTLper) infected mice. Whereas CTLac clones recognized a broad diversity of amino acid substitutions, CTLper clones exhibited exquisite specificity for the wild-type sequence. Highly focused specificity was CD8 independent but correlated with longer complementarity-determining regions 3 characteristic of CTLper clonotypes despite limited TCR alpha/beta-chain heterogeneity. Direct ex vivo analysis of CNS-derived mononuclear cells by IFN-gamma enzyme-linked immunospot assay confirmed the selection of T cells with narrow Ag specificity during persistence at the population level. These data suggest that broadly reactive CTL during primary infection are capable of controlling potentially emerging mutations. By contrast, the predominance of CD8+ T cells with dramatically focused specificity during persistence at the site of infection and in the periphery supports selective pressure driven by persisting Ag.     

Identification of K(+) channels in the plasma membrane of maize subsidiary cells

The stomatal complex of Zea mays consists of two guard cells with the pore in between them and two flanking subsidiary cells. Both guard cells and subsidiary cells are important elements for stoma physiology because a well-coordinated transmembrane shuttle transport of potassium and chloride ions occurs between these cells during stomatal movement. To shed light upon the corresponding transport systems from subsidiary cells, subsidiary cell protoplasts were enzymatically isolated and in turn, analyzed with the patch-clamp technique. Thereby, two K(+)-selective channel types were identified in the plasma membrane of subsidiary cells. With regard to their voltage-dependent gating behavior, they may act as hyperpolarization-dependent K(+) uptake and depolarization-activated K(+) release channels during stomatal movement. Interestingly, the K(+) channels from subsidiary cells and guard cells similarly responded to membrane voltage as well as to changes in the K(+) gradient. Further, the inward- and outward-rectifying K(+) current amplitude decreased upon a rise in the intracellular free Ca(2+) level from 2 nM to the micro M-range. The results indicate that the plasma membrane of subsidiary cells and guard cells has to be inversely polarized in order to achieve the anti-parallel direction of K(+) fluxes between these cell types during stomatal movement.     

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase,that mediates the first committed step in penicillin biosynthesis,is a cytosolic enzyme

Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-alpha-aminoadipate, L-cysteine, and L-valine into the tripeptide ACV. ACV synthetase has previously been localized to the vacuole where it is thought to utilize amino acids from the vacuolar pools. We localized ACV synthetase by subcellular fractionation and immuno-electron microscopy under conditions that prevented proteolysis and found it to co-localize with isopenicillin N synthetase in the cytosol, while acyltransferase localizes in microbodies. These data imply that the key enzymatic steps in penicillin biosynthesis are confined to only two compartments, i.e., the cytosol and microbody.