The significance of the hydrophilic backbone and the hydrophobic fatty acid regions of lipid a for macrophage binding and cytokine induction

Abstract Natural partial structures of lipopolysaccharide (LPS) as well as synthetic analogues and derivatives of lipid A were compared with respect to inhibit the binding of ¹²⁵I-labelled Re-chemotype LPS to mouse macrophage-like J774.1 cells to induce cytokine-release in J774.1 cells. LPS, synthetic Escherichia coli -type lipid A (compound 506) and tetraacyl percursor Ia (compound 406) inhibited the binding of ¹²⁵I-LPS to macrophage-like J774.1 cells and induced the release of tumor ncerosis factor α (TNFα) and interleukin 6 (IL-6). Deacylated R-chemotype LPS preparations were completely inactive in inhibiting binding and in inducing cytokine-release. Among tetraacyl compounds, the inhibition-capacity of LPS-binding was in decreasing order: PE-4 ( α -phosphonooxyethyl analogue of 406)>406⪢>404(4′-monophosphoryl partial structure of 406)>405 (1-monophosphoryl partial structure of 406). In the case of hexaccyl preparations, compounds 506, PE-1 (α-phosphonooxyethyl analogue of 506) and PE-2 (differing from PE-1 in having 14:0 at positions 2 and 3 of the reducing GlcN) inhibited LPS-binding and induced cytokine release equally well, whereas preparation PE-3 (differing from PE-2 in containing a β-phosphhonooxyethyl group) showed a substantially lower capacity in binding-inhibition and cytokine-induction. The conclusion is that chemical changes in the hydrophilic lipid A backbone reduce the capacity of lipid A to bind to cells, whereas the number of fatty acids determines the capacity of lipid A to activate cells. These results indicate that the bisphosphorylated hexosamine backbone of lipid A is essential for specific binding of LPS to macrophages and that the acylation pattern plays a critical role for LPS-promoted cell activation, i.e. cytokine induction.     

Methane production through anaerobic digestion of various energy crops grown in sustainable crop rotations

Biogas production is of major importance for the sustainable use of agrarian biomass as renewable energy source. Economic biogas production depends on high biogas yields. The project aimed at optimising anaerobic digestion of energy crops. The following aspects were investigated: suitability of different crop species and varieties, optimum time of harvesting, specific methane yield and methane yield per hectare. The experiments covered 7 maize, 2 winter wheat, 2 triticale varieties, 1 winter rye, and 2 sunflower varieties and 6 variants with permanent grassland. In the course of the vegetation period, biomass yield and biomass composition were measured. Anaerobic digestion was carried out in eudiometer batch digesters. The highest methane yields of 7500-10200 m(N)(3)ha(-1) were achieved from maize varieties with FAO numbers (value for the maturity of the maize) of 300 to 600 harvested at "wax ripeness". Methane yields of cereals ranged from 3200 to 4500 m(N)(3)ha(-1). Cereals should be harvested at "grain in the milk stage" to "grain in the dough stage". With sunflowers, methane yields between 2600 and 4550 m(N)(3)ha(-1) were achieved. There were distinct differences between the investigated sunflower varieties. Alpine grassland can yield 2700-3500 m(N)(3)CH(4)ha(-1). The methane energy value model (MEVM) was developed for the different energy crops. It estimates the specific methane yield from the nutrient composition of the energy crops. Energy crops for biogas production need to be grown in sustainable crop rotations. The paper outlines possibilities for optimising methane yield from versatile crop rotations that integrate the production of food, feed, raw materials and energy. These integrated crop rotations are highly efficient and can provide up to 320 million t COE which is 96% of the total energy demand of the road traffic of the EU-25 (the 25 Member States of the European Union).     

Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both CD44v6 and F-actin

In several types of cells, the activation of the receptor tyrosine kinase c-Met by its ligand hepatocyte growth factor (HGF) requires the coreceptor CD44v6. The CD44 extracellular domain is necessary for c-Met autophosphorylation, whereas the intracellular domain is required for signal transduction. We have already shown that the CD44 cytoplasmic tail recruits ezrin, radixin and moesin (ERM) proteins to the complex of CD44v6, c-Met, and HGF. We have now defined the function of the ERM proteins and the step they promote in the signaling cascade. The association of ERM proteins to the coreceptor is absolutely required to mediate the HGF-dependent activation of Ras by the guanine nucleotide exchange factor Sos. The ERM proteins need, in addition, to be linked to the actin cytoskeleton to catalyze the activation of Ras. Thus, we describe here a new function of the cytoskeleton. It is part of a "signalosome" complex that organizes the activation of Ras by Sos. So far the cytoskeleton has mainly been identified as a "responder" to signal transduction. Here, we show now that F-actin acts as an "inducer" that actively organizes the signaling cascade.     

Genetically different wine yeasts isolated from Austrian vine-growing regions influence wine aroma differently and contain putative hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii

To evaluate the influence of the genomic properties of yeasts on the formation of wine flavour, genotypic diversity among natural Saccharomyces cerevisiae strains originating from grapes collected in four localities of three Austrian vine-growing areas (Thermenregion: locations Perchtoldsdorf and Pfaffst?tten, Neusiedlersee-Hügelland: location Eisenstadt, Neusiedlersee: location Halbturn) was investigated and the aroma compounds produced during fermentation of the grape must of 'Grüner Veltliner' were identified. Amplified fragment length polymorphism analysis (AFLP) showed that the yeast strains cluster in four groups corresponding to their geographical origin. The genotypic analysis and sequencing of the D1/D2 domain of 26S rRNA encoding gene and ITS1/ITS2 regions indicated that the Perchtoldsdorf strains were putative interspecies hybrids between S. cerevisiae and Saccharomyces kudriavzevii. Analysis of the aroma compounds by GS/MS indicated a region-specific influence of the yeasts on the chemical composition of the wines. The aroma compound profiles generated by the Perchtoldsdorf strains were more related to those produced by the Pfaffst?tten strains than by the Eisenstadt and Halbturn strains. Similar to the Pfaffst?tten yeasts, the putative hybrid strains were good ester producers, suggesting that they may influence the wine quality favourably.     

Enantiomeric analysis of amino acids by using comprehensive two-dimensional gas chromatography

The chiral separation of amino acids (AA) derivatised with ethyl chloroformate by using comprehensive two-dimensional gas chromatography is reported. A commercially available enantioselective capillary column (Chirasil-l-Val) has been tested as first-dimension column. Two nonenantioselective stationary phases (BPX50 and BP1) with different column lengths were combined with the enantioselective column, which represent chiral/polar and chiral/low-polarity column sets, respectively. These column sets were evaluated to determine the most useful column combination to provide improved separation efficiency of enantioselective AA analysis. Separations of AA mixtures derivatised either as their N-trifluoroacetyl methyl esters or with methyl chloroformate, performed on a chiral/low-polarity column set, are also shown. The method was demonstrated for chiral analysis of AAs in different beer samples. The major AA in the beer samples was proline with amounts ranging from around 65-95% with minor contents of glycine and the l-enantiomers of alanine, valine, leucine, and isoleucine. Small amounts of d-alanine, at about 1, 1.5, and 15% were detected in the three samples.     

Functional and molecular characterization of voltage-gated sodium channels in uteri from nonpregnant rats

We investigated the function and expression of voltage-gated Na(+) channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca(2+)] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50-60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca(2+)](i), which were abolished by treatment with 0.1 microM TTX. Using end-point and real-time quantitative RT-PCR, we detected the presence of the VGSC alpha subunits Scn2a1, Scn3a, Scn5a, and Scn8a in the cDNA from longitudinal muscle. The mRNAs of the auxiliary beta subunits Scbn1b, Scbn2b, Scbn4b, and traces of Scn3b were also present. These data show for the first time that Scn2a1, Scn3a, Scn5a, and Scn8a, as well as all VGSC beta subunits are expressed in the longitudinal smooth muscle layer of the rat myometrium. In addition, our data show that TTX-sensitive VGSC are able to mediate phasic contractions maintained over long periods of time in the uteri of nonpregnant rats.     

Thermal Behaviour of Honeybees During Aggressive Interactions

We report here on the interrelationship of aggressive behaviour and thermoregulation in honeybees. Body temperature measurements were carried out without behavioural disturbance by infrared thermography. Guard bees, foragers, drones, and queens involved in aggressive interactions were always endothermic, i.e. had their flight muscles activated. Guards made differential use of their endothermic capacity. Mean thorax temperature was 34.2–35.1°C during examination of bees but higher during fights with wasps (37°C) or attack of humans (38.6°C). They usually cooled down when examining bees whereas examinees often heated up during prolonged interceptions (maximum >47°C). Guards neither adjusted their thorax temperature (and thus flight muscle function and agility) to that of examined workers, nor to that of drones, which were 2–7°C warmer. Guards examined cool bees (<33°C) longer than warmer ones, supporting the hypothesis that heating of examinees facilitates odour identification by guards, probably because of vapour pressure increase of semiochemicals with temperature. Guards in the core of aggressive balls clinged to the attacked insects to fix them and kill them by heat (maximum 46.5°C). Bees in the outer cluster layers resembled normal guards behaviourally and thermally. They served as active core insulators by heating up to 43.9°C. While balled wasps were cooler (maximum 42.5°C) than clinging guards balled bees behaved like examinees with maximum temperatures of 46.6°C, which further supports the hypothesis that the examinees heat up to facilitate odour identification.     

Ecological stoichiometry of indirect grazer effects on periphyton nutrient content

Ecological stoichiometry has been successful in enhancing our understanding of trophic interactions between consumer and prey species. Consumer and prey dynamics have been shown to depend on the nutrient composition of the prey relative to the nutrient demand of the consumer. Since most experiments on this topic used a single consumer species, little is known about the validity of stoichiometric constraints on trophic interactions across consumers and ecosystems. We conducted a quantitative meta-analysis on grazer–periphyton experiments to test (1) if benthic grazers have consistent effects on the nutrient composition of their prey, and (2) whether these effects can be aligned to the nutrient stoichiometry of grazer and periphyton, other environmental factors, or experimental constraints. Grazers significantly lowered periphyton C:N and C:P ratios, indicating higher N- and P-content of grazed periphyton across studies. Grazer presence on average increased periphyton N:P ratios, but across studies the effect size did not differ significantly from zero. The sign and strength of grazer effects on periphyton nutrient ratios was strongly dependent on the nutrient content of grazers and their food, but also on grazer biomass, the amount of biomass removal and water column nutrients. Grazer with low P-content tended to reduce periphyton P-content, whereas grazers with high P-content increased periphyton P-content. This result suggests that low grazer P-content can be an indication of physiological P-limitation rather than a result of having relatively low and fixed P-requirements. At the across-system scale of this meta-analysis, predictions from stoichiometric theory are corroborated, but the plasticity of the consumer nutrient composition has to be acknowledged. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biphasic regulation of HMG-CoA reductase expression and activity during wound healing and its functional role in the control of keratinocyte angiogenic and proliferative responses

In this study, we determined the regulation and potential function of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) during skin repair in mice. Upon skin injury, healthy mice exhibited a biphasic increase in HMGR expression and activity with elevated levels at days 3 and 13 post-wounding. In situ hybridization revealed wound margin keratinocytes as a cellular source of HMGR expression. In vitro experiments using cultured HaCaT keratinocytes uncovered epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, and insulin as potent co-inducers of HMGR activity and vascular endothelial growth factor (VEGF) in the cells. Insulin-, but not EGF-mediated VEGF protein expression was functionally connected to co-induced HMGR activity, as simvastatin restrictively interfered only with insulin-induced translation of VEGF mRNA by inhibition of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation. Functional ablation of insulin-induced sterol regulatory element-binding protein (SREBP)-2 by siRNA abolished HMGR expression and insulin-triggered VEGF protein release from keratinocytes. Simvastatin also blocked proliferation of cultured keratinocytes. The observed inhibitory effects of simvastatin on keratinocyte VEGF expression and proliferation could be reversed by mevalonate, the product of HMGR enzymatic activity. In accordance, simvastatin-mediated inhibition of HMGR activity in acutely regenerating tissue of wounded mice was paralleled by a marked loss of VEGF protein expression and disturbances of normal proliferation processes in wound margin keratinocytes during skin repair.     

Study of early leaf senescence in Arabidopsis thaliana by quantitative proteomics using reciprocal 14N/15N labeling and difference gel electrophoresis

Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senescence, we designed an elaborate double and reverse labeling strategy simultaneously employing fluorescent two-dimensional DIGE as well as metabolic (15)N labeling followed by MS. Reciprocal (14)N/(15)N labeling of entire Arabidopsis thaliana plants showed that full incorporation of (15)N into the proteins of the plant did not cause any adverse effects on development and protein expression. A direct comparison of DIGE and (15)N labeling combined with MS showed that results obtained by both quantification methods correlated well for proteins showing low to moderate regulation factors. Nano HPLC/ESI-MS/MS analysis of 21 protein spots that consistently exhibited abundance differences in nine biological replicates based on both DIGE and MS resulted in the identification of 13 distinct proteins and protein subunits that showed significant regulation in Arabidopsis mutant plants displaying advanced leaf senescence. Ribulose 1,5-bisphosphate carboxylase/oxygenase large and three of its four small subunits were found to be down-regulated, which reflects the degradation of the photosynthetic machinery during leaf senescence. Among the proteins showing higher abundance in mutant plants were several members of the glutathione S-transferase family class phi and quinone reductase. Up-regulation of these proteins fits well into the context of leaf senescence since they are generally involved in the protection of plant cells against reactive oxygen species which are increasingly generated by lipid degradation during leaf senescence. With the exception of one glutathione S-transferase isoform, none of these proteins has been linked to leaf senescence before.