Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria

Myxobacteria are common in terrestrial habitats and well known for their formation of fruiting bodies and production of secondary metabolites. We studied a cluster of myxobacteria consisting only of sequences of marine origin (marine myxobacteria cluster, MMC) in sediments of the North Sea. Using a specific PCR, MMC sequences were detected in North Sea sediments down to 2.2 m depth, but not in the limnetic section of the Weser estuary and other freshwater habitats. In the water column, this cluster was only detected on aggregates up to a few meters above the sediment surface, but never in the fraction of free-living bacteria. A quantitative real-time PCR approach revealed that the MMC constituted up to 13% of total bacterial 16S rRNA genes in surface sediments of the North Sea. In a global survey, including sediments from the Mediterranean Sea, the Atlantic, Pacific and Indian Ocean and various climatic regions, the MMC was detected in most samples and to a water depth of 4300 m. Two fosmids of a library from sediment of the southern North Sea containing 16S rRNA genes affiliated with the MMC were sequenced. Both fosmids have a single unlinked 16S rRNA gene and no complete rRNA operon as found in most bacteria. No synteny to other myxobacterial genomes was found. The highest numbers of orthologues for both fosmids were assigned to Sorangium cellulosum and Haliangium ochraceum. Our results show that the MMC is an important and widely distributed but largely unknown component of marine sediment-associated bacterial communities.     

Hyperthermophilic endoglucanase for in planta lignocellulose conversion

ABSTRACT: BACKGROUND: The enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass. RESULTS: Here we show that a cellulase gene (sso1354) isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade alpha-cellulose or even complex cell wall preparations under those pretreatment conditions. CONCLUSION: The hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications.     

Hypothalamic orexin--a neurons are involved in the response of the brain stress system to morphine withdrawal

Both the hypothalamus-pituitary-adrenal (HPA) axis and the extrahypothalamic brain stress system are key elements of the neural circuitry that regulates the negative states during abstinence from chronic drug exposure. Orexins have recently been hypothesized to modulate the extended amygdala and to contribute to the negative emotional state associated with dependence. This study examined the impact of chronic morphine and withdrawal on the lateral hypothalamic (LH) orexin A (OXA) gene expression and activity as well as OXA involvement in the brain stress response to morphine abstinence. Male Wistar rats received chronic morphine followed by naloxone to precipitate withdrawal. The selective OX1R antagonist SB334867 was used to examine whether orexins' activity is related to somatic symptoms of opiate withdrawal and alterations in HPA axis and extended amygdala in rats dependent on morphine. OXA mRNA was induced in the hypothalamus during morphine withdrawal, which was accompanied by activation of OXA neurons in the LH. Importantly, SB334867 attenuated the somatic symptoms of withdrawal, and reduced morphine withdrawal-induced c-Fos expression in the nucleus accumbens (NAc) shell, bed nucleus of stria terminalis, central amygdala and hypothalamic paraventricular nucleus, but did not modify the HPA axis activity. These results highlight a critical role of OXA signalling, via OX1R, in activation of brain stress system to morphine withdrawal and suggest that all orexinergic subpopulations in the lateral hypothalamic area contribute in this response.     

Impact of Land-Use Intensity and Productivity on Bryophyte Diversity in Agricultural Grasslands

While bryophytes greatly contribute to plant diversity of semi-natural grasslands, little is known about the relationships between land-use intensity, productivity, and bryophyte diversity in these habitats. We recorded vascular plant and bryophyte vegetation in 85 agricultural used grasslands in two regions in northern and central Germany and gathered information on land-use intensity. To assess grassland productivity, we harvested aboveground vascular plant biomass and analyzed nutrient concentrations of N, P, K, Ca and Mg. Further we calculated mean Ellenberg indicator values of vascular plant vegetation. We tested for effects of land-use intensity and productivity on total bryophyte species richness and on the species richness of acrocarpous (small & erect) and pleurocarpous (creeping, including liverworts) growth forms separately. Bryophyte species were found in almost all studied grasslands, but species richness differed considerably between study regions in northern Germany (2.8 species per 16 m²) and central Germany (6.4 species per 16 m²) due environmental differences as well as land-use history. Increased fertilizer application, coinciding with high mowing frequency, reduced bryophyte species richness significantly. Accordingly, productivity estimates such as plant biomass and nitrogen concentration were strongly negatively related to bryophyte species richness, although productivity decreased only pleurocarpous species. Ellenberg indicator values for nutrients proved to be useful indicators of species richness and productivity. In conclusion, bryophyte composition was strongly dependent on productivity, with smaller bryophytes that were likely negatively affected by greater competition for light. Intensive land-use, however, can also indirectly decrease bryophyte species richness by promoting grassland productivity. Thus, increasing productivity is likely to cause a loss of bryophyte species and a decrease in species diversity.     

Characterization of HLA class II/peptide-TCR interactions of the immunodominant T cell epitope in Art v 1, the major mugwort pollen allergen

More than 95% of mugwort pollen-allergic individuals are sensitized to Art v 1, the major allergen in mugwort pollen. Interestingly, the CD4 T cell response to Art v 1 involves only one single immunodominant peptide, Art v 1(25-36) (KCIEWEKAQHGA), and is highly associated with the expression of HLA-DR1. Therefore, we investigated the molecular basis of this unusual immunodominance among allergens. Using artificial APC expressing exclusively HLA-DRB1*0101 and HLA-DRA*0101, we formally showed that DR1 acts as restriction element for Art v 1(25-36)-specific T cell responses. Further assessment of binding of Art v 1(25-36) to artificial HLA-DR molecules revealed that its affinity was high for HLA-DR1. Amino acid I27 was identified as anchor residue interacting with DR molecules in pocket P1. Additionally, Art v 1(25-36) bound with high affinity to HLA-DRB1*0301 and *0401, moderately to HLA-DRB1*1301 and HLA-DRB5*0101, and weakly to HLA-DRB1*1101 and *1501. T cell activation was also inducible by Art v 1(25-36)-loaded, APC-expressing HLA molecules other than DR1, indicating degeneracy of peptide binding and promiscuity of TCR recognition. Specific binding of HLA-DRB1*0101 tetramers containing Art v 1(19-36) allowed the identification of Art v 1(25-36)-specific T cells by flow cytometry. In summary, the immunodominance of Art v 1(25-36) relies on its affinity to DR1, but is not dictated by it. Future investigations at the molecular HLA/peptide/TCR and cellular level using mugwort pollen allergy as a disease model may allow new insights into tolerance and pathomechanisms operative in type I allergy, which may instigate new, T cell-directed strategies in specific immunotherapy.     

Bioactivity of folding intermediates studied by the recovery of enzymatic activity during refolding

The peptide bond preceding proline residues realizes a cis/trans conformational switch with high switching resistance in native proteins and folding intermediates. Therefore, individual isomers have the potential to differ in bioactivity. However, information about isomer-specific bioactivities is difficult to obtain because of the risk of affecting isomeric distribution by bioactivity assay components.Here we present an approach that allows for the measurement of the recovery of enzymatic activities of wild-type RNase T₁ and RNase T₁ variants during refolding under conditions where the population of enzyme-substrate or enzyme-product complexes is negligible. Recovery of enzymatic activity was continuously monitored within the visible range of the spectrum by addition of a fluorescence-labeled nucleotide substrate to the refolding sample. We found that a nonnative trans conformation at Pro39 renders the RNase T₁ almost completely inactive. A folding intermediate having a nonnative trans conformation at Pro55 shows about 46% of the enzymatic activity referred to the native state. Pro55, in contrast to the active site located Pro39, is situated in a solvent-exposed loop region remote from active-site residues. In both cases, peptidyl prolyl cis/trans isomerases accelerate the regain of nucleolytic activity. Our findings show that even if there is a considerable distance between the site of isomerization and the active site, conformational control of the bioactivity of proteins is likely to occur, and that the surface location of prolyl bonds suffices for the control of buried active sites mediated by peptidyl prolyl cis/trans isomerases.     

Neutropenia with impaired immune response to Streptococcus pneumoniae in ceramide kinase-deficient mice

In mammals, ceramide kinase (CerK)-mediated phosphorylation of ceramide is the only known pathway to ceramide-1-phosphate (C1P), a recently identified signaling sphingolipid metabolite. To help delineate the roles of CerK and C1P, we knocked out the gene of CerK in BALB/c mice by homologous recombination. All in vitro as well as cell-based assays indicated that CerK activity is completely abolished in Cerk-/- mice. Labeling with radioactive orthophosphate showed a profound reduction in the levels of de novo C1P formed in Cerk-/- macrophages. Consistently, mass spectrometry analysis revealed a major contribution of CerK to the formation of C16-C1P. However, the significant residual C1P levels in Cerk-/- animals indicate that alternative routes to C1P exist. Furthermore, serum levels of proapoptotic ceramide in these animals were significantly increased while levels of dihydroceramide as the biosynthetic precursor were reduced. Previous literature pointed to a role of CerK or C1P in innate immune cell function. Using a variety of mechanistic and disease models, as well as primary cells, we found that macrophage- and mast cell-dependent readouts are barely affected in the absence of CerK. However, the number of neutrophils was strikingly reduced in blood and spleen of Cerk-/- animals. When tested in a model of fulminant pneumonia, Cerk-/- animals developed a more severe disease, lending support to a defect in neutrophil homeostasis following CerK ablation. These results identify ceramide kinase as a key regulator of C1P, dihydroceramide and ceramide levels, with important implications for neutrophil homeostasis and innate immunity regulation.     

Cutting edge: central nervous system plasmacytoid dendritic cells regulate the severity of relapsing experimental autoimmune encephalomyelitis

Plasmacytoid dendritic cells (pDCs) have both stimulatory and regulatory effects on T cells. pDCs are a major CNS-infiltrating dendritic cell population during experimental autoimmune encephalomyelitis but, unlike myeloid dendritic cells, have a minor role in T cell activation and epitope spreading. We show that depletion of pDCs during either the acute or relapse phases of experimental autoimmune encephalomyelitis resulted in exacerbation of disease severity. pDC depletion significantly enhanced CNS but not peripheral CD4(+) T cell activation, as well as IL-17 and IFN-gamma production. Moreover, CNS pDCs suppressed CNS myeloid dendritic cell-driven production of IL-17, IFN-gamma, and IL-10 in an IDO-independent manner. The data demonstrate that pDCs play a critical regulatory role in negatively regulating pathogenic CNS CD4(+) T cell responses, highlighting a new role for pDCs in inflammatory autoimmune disease.     

MLC-kinase/phosphatase control of Ca2+ signal transduction in airway smooth muscles

In airway smooth muscles, kinase/phosphatase-dependent phosphorylation and dephosphorylation of the myosin light chain (MLC) have been revealed by many authors as important steps in calcium (Ca²⁺) signalling pathway from the variation of Ca²⁺ concentration in cytosol to the force development. Here, a theoretical analysis of the control action of MLC-kinase (MLCK) and MLC-phosphatase (MLCP) in Ca²⁺ signalling is presented and related to the general control principles of these enzymes, which were previously studied by Reinhart Heinrich and his co-workers. The kinetic scheme of the mathematical model considers interactions among Ca²⁺, calmodulin (CaM) and MLCK and the well-known 4-state actomyosin latch bridge model, whereby a link between them is accomplished by the conservation relation of all species of MLCK. The mathematical model predicts the magnitude and velocity of isometric force in smooth muscles upon transient biphasic Ca²⁺ signal. The properties of signal transduction in the system such as the signalling time, signal duration and signal amplitude, which are reflected in the properties of force developed, are studied by the principles of the metabolic control theory. The analysis of our model predictions confirms as shown by Reinhart Heinrich and his co-workers that MLCK controls the amplitude of signal more than its duration, whereas MLCP controls both. Finally, the simulations of elevated total content of MLCK, a typical feature of bronchial muscles of asthmatic subjects and spontaneously hypertensive rats as well as potentiation of MLCP catalytic activity, are carried out and are discussed in view of an increase in the force magnitude.