Murein Synthesis and Identification of Cell Wall Precursors of Temperature-Sensitive Lysis Mutants of Escherichia coli

A group of temperature-sensitive lysis mutants of Escherichia coli K-12 was studied. Mutants impaired in the synthesis of uridine diphosphate-N-acetylmuramyl (UDP-MurNAc)-pentapeptide or in the synthesis of murein amino acids were found. Their rate of murein synthesis at the restrictive temperature was decreased. A large number of mutants did not differ from the parent strain with respect to the rate of murein synthesis and the precursor pattern. The behavior of these mutants is discussed. It was impossible to accumulate UDP-MurNAc-pentapeptide in E. coli by the antibiotics penicillin and vancomycin. The hypothesis is put forward that the amount of this murein precursor is regulated by feedback inhibition.     

Production of tailor made short chain amylose–lipid complexes using varying reaction conditions

When amylose was synthesized using potato phosphorylase in the presence of amylose complexing lipids, monodisperse populations of amylose–lipid complexes were formed. Enzyme dosage and glucose-1-phosphate (glc-1-P)/primer ratio influenced the reaction rate of the enzymic synthesis, presumably by changing the balance between amylose synthesis and amylose–lipid complexation and precipitation, and impacted the molecular weight of the complexes. Lipid characteristics affected the dissociation properties and amylose chain lengths of the amylose–lipid complexes presumably by determining the minimal amylose chain length necessary for complexation and precipitation. Tailor made short chain amylose–lipid complexes can hence be produced by choosing the appropriate reaction conditions. We propose a synthesis mechanism in which the primer is elongated until an amylose chain is obtained which is of sufficient length to complex a first lipid. Further chain extension then occurs, together with subsequent complexation until the complex becomes insoluble and precipitates.     

bioRad: biological analysis and visualization of weather radar data

Weather surveillance radars are increasingly used for monitoring the movements and abundances of animals in the airspace. However, analysis of weather radar data remains a specialised task that can be technically challenging. Major hurdles are the difficulty of accessing and visualising radar data on a software platform familiar to ecologists and biologists, processing the low‐level data into products that are biologically meaningful, and summarizing these results in standardized measures. To overcome these hurdles, we developed the open source R package bioRad, which provides a toolbox for accessing, visualizing and analyzing weather radar data for biological studies. It provides functionality to access low‐level radar data, process these data into meaningful biological information on animal speeds and directions at different altitudes in the atmosphere, visualize these biological extractions, and calculate further summary statistics. The package aims to standardize methods for extracting and reporting biological signals from weather radars. Here we describe a roadmap for analyzing weather radar data using bioRad. We also define weather radar equivalents for familiar measures used in the field of migration ecology, such as migration traffic rates, and recommend several good practices for reporting these measures. The bioRad package integrates with low‐level data from both the European radar network (OPERA) and the radar network of the United States (NEXRAD). bioRad aims to make weather radar studies in ecology easier and more reproducible, allowing for better inter‐comparability of studies.     

Kernel-based data fusion for gene prioritization

MOTIVATION: Hunting disease genes is a problem of primary importance in biomedical research. Biologists usually approach this problem in two steps: first a set of candidate genes is identified using traditional positional cloning or high-throughput genomics techniques; second, these genes are further investigated and validated in the wet lab, one by one. To speed up discovery and limit the number of costly wet lab experiments, biologists must test the candidate genes starting with the most probable candidates. So far, biologists have relied on literature studies, extensive queries to multiple databases and hunches about expected properties of the disease gene to determine such an ordering. Recently, we have introduced the data mining tool ENDEAVOUR (Aerts et al., 2006), which performs this task automatically by relying on different genome-wide data sources, such as Gene Ontology, literature, microarray, sequence and more. RESULTS: In this article, we present a novel kernel method that operates in the same setting: based on a number of different views on a set of training genes, a prioritization of test genes is obtained. We furthermore provide a thorough learning theoretical analysis of the method's guaranteed performance. Finally, we apply the method to the disease data sets on which ENDEAVOUR (Aerts et al., 2006) has been benchmarked, and report a considerable improvement in empirical performance. AVAILABILITY: The MATLAB code used in the empirical results will be made publicly available.     

Corticosterone levels in host and parasite nestlings: Is brood parasitism a hormonal stressor?

Parasite chicks from non-evictor species usually try to monopolize host parental care, thereby increasing considerably the level of food competition in the nest. Here, we propose that brood parasitism is an important stressor for host and parasite nestlings and explore this hypothesis in the non-evictor great spotted cuckoo (Clamator glandarius) and its main hosts, the same-sized black-billed magpie (Pica pica) and the larger carrion crow (Corvus corone). We experimentally created 3-nestling broods of different brood compositions (only cuckoo chicks, only host chicks, or cuckoo and host chicks together) and measured baseline corticosterone levels of nestlings along their developmental period (early, middle and late). We found that brood parasitism increased corticosterone levels in magpie nestlings in the mid and late nestling period compared to those raised in unparasitized nests. Interestingly, carrion crow nestlings from parasitized nests only increased their corticosterone levels in the mid nestling period, when the competition for food with the cuckoo nestling was highest. Our results suggest that brood parasitism could be a potential physiological stressor for host nestlings, especially during the developmental stages where food requirements are highest. Conversely, cuckoo nestlings could be physiologically adapted to high competition levels since they did not show significant differences in corticosterone levels in relation to brood composition.     

Diffusion Limitations in Root Uptake of Cadmium and Zinc, But Not Nickel, and Resulting Bias in the Michaelis Constant

It has long been recognized that diffusive boundary layers affect the determination of active transport parameters, but this has been largely overlooked in plant physiological research. We studied the short-term uptake of cadmium (Cd), zinc (Zn), and nickel (Ni) by spinach (Spinacia oleracea) and tomato (Lycopersicon esculentum) in solutions with or without metal complexes. At same free ion concentration, the presence of complexes, which enhance the diffusion flux, increased the uptake of Cd and Zn, whereas Ni uptake was unaffected. Competition effects of protons on Cd and Zn uptake were observed only at a very large degree of buffering, while competition of magnesium ions on Ni uptake was observed even in unbuffered solutions. These results strongly suggest that uptake of Cd and Zn is limited by diffusion of the free ion to the roots, except at very high degree of solution buffering, whereas Ni uptake is generally internalization limited. All results could be well described by a model that combined a diffusion equation with a competitive Michaelis-Menten equation. Direct uptake of the complex was estimated to be a major contribution only at millimolar concentrations of the complex or at very large ratios of complex to free ion concentration. The true K_m for uptake of Cd²⁺ and Zn²⁺ was estimated at <5 nm, three orders of magnitude smaller than the K_m measured in unbuffered solutions. Published Michaelis constants for plant uptake of Cd and Zn likely strongly overestimate physiological ones and should not be interpreted as an indicator of transporter affinity.Internalization of metals by biota is traditionally described by Michaelis-Menten kinetics (Wilkinson and Buffle, 2004). The K_m corresponds to the concentration in solution at which the uptake is one-half of the maximal uptake, F_max. The Michaelis-Menten equation relates the uptake flux, F, to the free ion concentration at the site of uptake, [M]_s:If diffusion of a metal across a diffusive boundary layer adjacent to the roots is the rate-limiting step for uptake, the concentration at the site of uptake will be lower than that in the bulk solution. As a result, diffusion limitations result in an overestimate of the K_m, if the concentration at the root surface is assumed to be the same as in the bulk solution, as is usually done. This bias in K_m has been discussed in detail by Winne (1973) and has, for instance, been demonstrated experimentally for uptake of Glc in rabbit jejunum (Thomson and Dietschy, 1980) and for uptake of several sugars, amino acids, and bile acids in rat ileum (Wilson and Dietschy, 1974).Models used to predict ion availability and toxicity of metals by plants usually rely on the assumption that uptake is controlled by the free metal ion activity and the activity of competing ions in the bulk solution. For instance, the biotic ligand model (BLM), originally developed to predict metal toxicity to aquatic organisms, assumes that toxicity of an ion is mitigated by the presence of competing ions that bind on the biotic ligand (Paquin et al., 2002). Hough et al. (2005) used a free ion activity model taking into account proton competition effects to predict cadmium (Cd) uptake by soil-grown ryegrass (Lolium perenne). The uptake was reasonably well predicted; however, as the authors pointed out, it was not clear whether the derived constants truly represented physiological affinity constants or were just fitting parameters in a rate-limited uptake process. In case of strong diffusion limitation, ion competition effects on the internalization are expected to have negligible effect on the uptake, as the uptake is controlled by diffusion and not by internalization (Campbell et al., 2002; Degryse and Smolders, 2012).In previous studies, we found strong evidence that uptake of Cd and zinc (Zn) is limited by the diffusive transport of the free metal ion to the root at low free ion concentration. At constant free ion concentration, the uptake of Cd and Zn increased in presence of metal complexes and the contribution of the complex increased with increasing dissociation rate of the complex (Degryse et al., 2006a, 2006c). In unbuffered solutions, i.e. solutions without metal complexes, stirring increased Cd uptake by plants (Degryse and Smolders, 2012). For nickel (Ni), however, contribution of complexes was small or undetectable, and stirring did not increase the uptake (Degryse and Smolders, 2012). Given this evidence that Cd and Zn uptake by plants is limited by diffusion, it is likely that published K_m values for uptake of Cd²⁺ and Zn²⁺ by plants overestimate true physiological values. This bias in the K_m when a diffusive boundary layer is present has been largely ignored in plant-physiological research. Indeed, in numerous studies the K_m value has been interpreted as a characteristic of the carrier-mediated transport process, while in many cases it may reflect mass transfer properties. In addition, these diffusion limitations may mask ion competition effects in the uptake.The aim of this article was (1) to present evidence that K_m values determined for Cd²⁺ and Zn²⁺ uptake by plants in general reflect transport limitations rather than transporter affinity; (2) to derive true K_m values by determining the K_m under conditions where the uptake is not transport limited; (3) to identify the consequence of diffusion limitations on competition effects; and (4) to describe uptake of Cd, Zn, and Ni by plants in a single comprehensive model that combines competitive Michaelis-Menten kinetics with a diffusion equation.

Theoretical Framework

In the following, we qualitatively discuss the bias in the K_m because of diffusion limitations, based on Figure 1. Equations are given in the “Materials and Methods” section. Figure 1A presents a case where the potential internalization flux by the plant at low concentrations is much larger than the maximal rate at which the free ion can be supplied through diffusive transport of the ion to the root surface. In this case, the actual uptake flux by the plant will approach the maximal diffusive flux, and the free ion concentration at the root surface is much smaller than that in the bulk solution. The apparent K_m (K_m*; determined as the concentration where the uptake flux is one-half of the maximal uptake) is much larger than the true K_m value. In Figure 1B, the potential internalization flux at low concentration is of the same order of magnitude as the maximal diffusion flux. In this case, the concentration at the root surface is slightly smaller than that in the bulk solution, and there is only a slight bias in the K_m value.Open in a separate windowFigure 1.Conceptual diagram of the internalization flux (Michaelis-Menten curve; full line), maximal diffusive supply from solution to root (dashed line), and actual uptake flux (dotted line) as a function of free ion concentration for two theoretical cases. Left and right sections show the same curves, on log (left) or linear (right) scale. In A, the potential internalization flux is much larger than the maximal diffusive supply at low concentrations, i.e. the uptake is strongly limited by the transport of the free ion to the root. The plant acts as a near-zero sink, and the actual plant uptake equals the maximal diffusive flux. The K_m* is much larger than the true K_m, and the experimental permeability P (slope of the actual uptake curve) is much smaller than the membrane permeability P_m (slope of the internalization curve). In B, the maximal diffusive flux is larger than the potential internalization flux. The uptake is not limited by diffusive transport, and the K_m* and true K_m are almost equal.Given the evidence that uptake of Cd and Zn by plants is diffusion limited, even in stirred nutrient solutions, we hypothesize that reported K_m values of Cd²⁺ and Zn²⁺ are biased, and that the physiological K_m values are much smaller. To test this hypothesis, we measured the uptake of Cd, Zn, and Ni in solution, in absence or in presence of labile hydrophilic metal complexes. If diffusion limitations prevail, the complexes dissociate within the diffusion layer and thus enhance the diffusion flux and therefore the metal uptake. By adding labile complexes in large amounts, it should be possible to abolish the diffusion barrier completely, in which case the physiological K_m can be determined.In addition, the effect of competitive ions on uptake was tested. The presence of competitive ions decreases the internalization flux. However, if uptake is rate limited by the diffusive transport to the uptake site and not by internalization, competition effects should theoretically not affect the uptake flux.     

Fungal decay resistance and durability of organosilicon-treated wood

The potential use of organosilicons as protective agents against basidiomycetes attack of wood used in outdoor applications was investigated using Scots pine sapwood and beech specimens. Both mini-blocks and EN 113 specimens were subjected to brown-rot and white-rot fungi. A dose–response could be observed showing that with higher weight percentage gain of the organosilicon, the resistance (i.e., efficacy) against fungi increased. At relatively low weight percentage gains, which are assumed to be economically feasible, Scots pine could be partly protected against decay by Postia placenta and Coniophora puteana and beech could be partly protected against decay by C. puteana and Trametes versicolor. Full protection was achieved by some silicons for Scots pine sapwood against C. puteana and for beech against T. versicolor. The most promising products were a solvent-based mixture of the alkoxysilanes methyltrimethoxysilane (MTM) and octyltriethoxysilane (OTES) and a water-based micro-emulsion of polydimethylsiloxane (PDMS) and triethoxysilane (TES) when applied above 20 and 30% weight gain for Scots pine and above 30 and 40% weight gain for beech. A water-based mixture of dimethylmethylhydrogen siloxane (DMS) and N-octyltriethoxysilane (n-OTES) was able to protect beech at weight gains above 30%.     

Human mast cells express leptin and leptin receptors

Mast cells are immune cells that produce and secrete a variety of mediators and cytokines that influence various inflammatory and immune processes. Leptin is a cytokine regulating metabolic, endocrine as well as immune functions via the leptin receptor which is expressed by many immune cells. However, there are no data about leptin receptor expression in mast cells. Immunohistochemical and immunofluorescent double stainings showed the expression of leptin and leptin receptors in mast cells in human skin and several parts of the respiratory, gastrointestinal and urogenital tract. Leptin was expressed in mast cells expressing the classification marker chymase, whereas a variable expression was observed in tryptase positive mast cells. For leptin receptors, the expression pattern was tissue dependent and not related to tryptase or chymase expression. Our results demonstrate the expression of leptin and leptin receptors on mast cells, suggesting paracrine and/or autocrine immunomodulatory effects of leptin on mast cells.     

Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A

A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized.