Fractionation of three different lignins by thermal separation techniques—A comparative study

Separation of the heterogeneous lignin macromolecule in fractions with increased homogeneity, as well as different structural (molar mass) and functional (hydroxy groups, ‐OH) features is important in terms of its use as a chemical building block. For this purpose, three thermal separation techniques were investigated and compared: solvent extraction, successive precipitation and ultrafiltration. One important issue in this context is the utilization of organic solvents with low boiling points to ensure a simple and efficient recovery. In addition to a softwood Kraft lignin (Indulin AT) as reference lignin, two sulfur‐free Organosolv lignins from short rotation coppice (“poplar with bark”) and from the energy grass Miscanthus × gigantheus were investigated. The lignins were separated into low, medium and high molecular weight fractions. Due to the different initial structural features and the associated varying solubility properties in such lignins, different organic solvents were needed for dissolution and precipitation of the individual lignin fractions. The polarity of the used solvent is one key factor regarding the yield of the soluble fraction and the success of molecular sorting into low, medium, and high molecular weight. Further structural features, for example the aliphatic OH‐group content increased with rising molecular weight of poplar, miscanthus, and Kraft lignin from minimum 0.72, 0.3, and 1.6 mmol/g to maximum 2.4, 1.6, and 2.8 mmol/g, respectively. The number of phenolic OH‐groups decreased from maximum 3.8, 4.3, and 4.2 to minimum 1.4, 2.7, and 2.9, respectively. The presented work illustrate options regarding the molecular sorting of several lignin types with three thermal techniques into fractions differing in key properties (yield, molecular weight, polydispersity, functional groups) for material applications.     

AtDUR3 encodes a new type of high-affinity urea/H+ symporter in Arabidopsis

Urea is the major nitrogen form supplied as fertilizer in agricultural plant production but also an important nitrogen metabolite in plants. We report the cloning and functional characterization of AtDUR3, a high-affinity urea transporter in plants. AtDUR3 contains 14 putative transmembrane-spanning domains and represents an individual member in Arabidopsis that belongs to a superfamily of sodium-solute symporters. Heterologous expression in urea uptake-defective yeast as well as two-electrode voltage clamp and uptake studies using (14)C-labeled urea in AtDUR3-expressing oocytes demonstrated that AtDUR3 mediates urea transport. In both heterologous systems, urea transport was stimulated at low pH. In oocytes, inward currents indicated that urea is cotransported with protons. By contrast, a supply of Na(+) ions could not stimulate urea transport. Transport of (14)C-labeled urea by AtDUR3 in oocytes exhibited saturation kinetics with a K(m) of approximately 3 micro M. AtDUR3 was expressed in shoots and roots and upregulated during early germination and under nitrogen deficiency in roots. We propose a role of AtDUR3 in urea uptake by plant cells at low external urea concentrations.     

Does coping style predict optimization? An experimental test in a wild passerine bird

A number of studies have suggested that avian brood size is individually optimized. Yet, optimal reproductive decisions likely vary owing to among-individual differences in environmental sensitivity. Specifically, ‘proactive’ individuals who do not track environmental changes may be less able to produce optimal brood sizes than ‘reactive’ individuals who have more precise local environmental knowledge. To test this, we quantified exploratory behaviour (a proxy for proactivity) in a great tit (Parus major) population, manipulated brood sizes (reduced, control, enlarged) and evaluated whether individuals of dissimilar coping style differed in their level of optimization. If reactive females behaved optimally, any deviation from their original brood size should lower fitness, whereas this should not be the case for proactive females. Reactive females indeed performed best at their natural brood size, whereas proactive females performed best when raising an enlarged brood. These findings imply that proactive females produced sub-optimal brood sizes. We speculate that proactive females might (i) take decisions based on biased perception of their environment, (ii) face energetic constraints in offspring production and/or (iii) be more willing to invest into current reproduction when given the option. Our findings provide experimental evidence for coping style-related differences in optimal reproductive decisions and life-history strategies.     

Purification,biochemical characterization and gene sequencing of a thermostable raw starch digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov.

This study reports the purification and biochemical characterization of a raw starch-digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov. (strain Pizzo^T). The molecular weight was estimated to be 58 kDa by SDS–PAGE. The enzyme was highly active over a wide range of pH from 4.0–10.0. The optimum temperature of the enzyme was 70°C. It showed extreme thermostability in the presence of Ca²⁺, retaining 50% of its initial activity after 90 h at 70°C. The enzyme efficiently hydrolyzed 20% (w/v) of raw starches, concentration normally used in starch industries. The α-amylase showed an high stability in presence of many organic solvents. In particular the residual activity was of 73% in presence of 15% (v/v) ethyl alcohol, which corresponds to ethanol yield in yeast fermentation process. By analyzing its complete amyA gene sequence (1,542 bp), the enzyme was proposed to be a new α-amylase.     

Flocculating performances of exopolysaccharides produced by a halophilic bacterial strain cultivated on agro-industrial waste

This study reports the first systematic investigation of the flocculation dynamics of exopolysaccharides (EPSs) produced by a halophilic bacterial strain grown on pretreated molasses as fermentation substrate. The potential use of these EPSs as an easily biodegradable, natural alternative for synthetic polyelectrolytes which are widely used and contain toxic and carcinogenic monomers was investigated. Flocculating activities of the EPS samples in synthetic water, synthetic sea water and natural sea water media which were used as model raw waters were monitored via the Photometric Dispersion Analyser (PDA 2000) instrument and removals were determined by measuring residual turbidities. One of the six EPS specimens, which formed the largest flocs thus performed highest turbidity removal, exhibited flocculation performance and particle removal efficiency comparable with commercial cationic, nonionic and anionic synthetic polyelectrolytes.     

Effect of Oxidative Stress on the Junctional Proteins of Cultured Cerebral Endothelial Cells

Summary 1. There is increasing evidence that the cerebral endothelium and the blood–brain barrier (BBB) plays an important role in the oxidative stress-induced brain damage. The aim of the present study was to investigate the role of interendothelial junctional proteins in the BBB permeability increase induced by oxidative stress.2. For the experiments, we have used cultured cerebral endothelial cells exposed to hypoxia/reoxygenation or treated with the redox cycling quinone 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ) in the presence or absence of glucose. The expression of junctional proteins and activation of mitogen activated protein kinases (MAPK) was followed by Western-blotting, the interaction of junctional proteins was investigated using coimmunoprecipitation.3. Oxidative stress induces a downregulation of the tight junction protein occludin expression which is more pronounced in the absence of glucose. Furthermore, oxidative stress leads to disruption of the cadherin--catenin complex and an activation of extracellular signal-regulated kinase (ERK1/2), which is more intense in the absence of glucose.4. We have shown that one of the causes of the BBB breakdown is probably the structural alteration of the junctional complex caused by oxidative stress, a process in which ERK1/2 may play an important role.This revised article was published online in May 2005 with a February 2005 cover date.     

Tagging genes with cassette-exchange sites

In an effort to make transgenesis more flexible and reproducible, we developed a system based on novel 5′ and 3′ ‘gene trap’ vectors containing heterospecific Flp recognition target sites and the corresponding ‘exchange’ vectors allowing the insertion of any DNA sequence of interest into the trapped locus. Flp-recombinase-mediated cassette exchange was demonstrated to be highly efficient in our system, even in the absence of locus-specific selection. The feasibility of constructing a library of ES cell clones using our gene trap vectors was tested and a thousand insertion sites were characterized, following electroporation in ES cells, by RACE–PCR and sequencing. We validated the system in vivo for two trapped loci in transgenic mice and demonstrated that the reporter transgenes inserted into the trapped loci have an expression pattern identical to the endogenous genes. We believe that this system will facilitate in vivo studies of gene function and large-scale generation of mouse models of human diseases, caused by not only loss but also gain of function alleles.