Soil Microbial Diversity in the Vicinity of Desert Shrubs

Water and nutrient availability are the major limiting factors of biological activity in arid and semiarid ecosystems. Therefore, perennial plants have developed different ecophysiological adaptations to cope with harsh conditions. The chemical profile of the root exudates varies among plant species and this can induce variability in associated microbial populations. We examined the influence of two shrubs species, Artemisia sieberi and Noaea mucronata, on soil microbial diversity. Soil samples were collected monthly, from December 2006 to November 2007, near canopies of both shrubs (0–10-cm depth). Samples were used for abiotic tests and determination of soil bacterial diversity. No significant differences were found in the abiotic variables (soil moisture, total organic matter, and total soluble nitrogen (TSN)) between soil samples collected from under the two shrubs during the study period. No obvious differences in the Shannon–Weaver index, evenness values, or total phylogenetic distances were found for the soil microbial communities. However, detailed denaturing gradient gel electrophoresis (DGGE) clustering as well as taxonomic diversity analyses indicated clear shifts in the soil microbial community composition. These shifts were governed by seasonal variability in water availability and, significantly, by plant species type.     

Changes in tyrosinase specificity by ionic liquids and sodium dodecyl sulfate

Tyrosinase is a member of the type 3 copper enzyme family involved in the production of melanin in a wide range of organisms. The ability of tyrosinases to convert monophenols into diphenols has stimulated studies regarding the production of substituted catechols, important intermediates for the synthesis of pharmaceuticals, agrochemicals, polymerization inhibitors, and antioxidants. Despite its enormous potential, the use of tyrosinases for catechol synthesis has been limited due to the low monophenolase/diphenolase activity ratio. In the presence of two water miscible ionic liquids, [BMIM][BF₄] and ethylammonium nitrate, the selectivity of a tyrosinase from Bacillus megaterium (TyrBm) was altered, and the ratio of monophenolase/diphenolase activity increased by up to 5-fold. Furthermore, the addition of sodium dodecyl sulphate (SDS) at levels of 2–50 mM increased the activity of TyrBm by 2-fold towards the natural substrates l-tyrosine and l-Dopa and 15- to 20-fold towards the non-native phenol and catechol. The R209H tyrosinase variant we previously identified as having a preferential ratio of monophenolase/diphenolase activity was shown to have a 45-fold increase in activity towards phenol in the presence of SDS. We propose that the effect of SDS on the ability of tyrosinase to convert non-natural substrates is due to the interaction of surfactant molecules with residues located at the entrance to the active site, as visualized by the newly determined crystal structure of TyrBm in the presence of SDS. The effect of SDS on R209 may enable less polar substrates such as phenol and catechol, to penetrate more efficiently into the enzyme catalytic pocket.     

A Simple Method for Assessment of Human Anti-Neu5Gc Antibodies Applied to Kawasaki Disease

N-Glycolylneuraminic acid (Neu5Gc) is an immunogenic sugar of dietary origin that metabolically incorporates into diverse native glycoconjugates in humans. Anti-Neu5Gc antibodies are detected in all human sera, though with variable levels and epitope-recognition profiles. These antibodies likely play a role in several inflammation-mediated pathologies including cardiovascular diseases and cancer. In cancer, they have dualistic and opposing roles, either stimulating or repressing disease, as a function of their dose, and some of these antibodies serve as carcinoma biomarkers. Thus, anti-Neu5Gc antibodies may signify risk of inflammation-mediated diseases, and changes in their levels could potentially be used to monitor disease progression and/or response to therapy. Currently, it is difficult to determine levels of anti-Neu5Gc antibodies in individual human samples because these antibodies recognize multiple Neu5Gc-epitopes. Here we describe a simple and specific method for detection and overall estimation of human anti-Neu5Gc antibodies. We exploit the difference between two mouse models that differ only by Neu5Gc-presence (wild-type) or Neu5Gc-absence (Cmah^−/− knockout). We characterize mouse serum from both strains by HPLC, lectin and mass-spectrometry analysis and show the target Neu5Gc-epitopes. We then use Cmah^−/− knockout sera to inhibit all non-Neu5Gc-reactivity followed by binding to wild-type sera to detect overall anti-Neu5Gc response in a single assay. We applied this methodology to characterize and quantify anti-Neu5Gc IgG and IgA in sera of patients with Kawasaki disease (KD) at various stages compared to controls. KD is an acute childhood febrile disease characterized by inflammation of coronary arteries that untreated may lead to coronary artery aneurysms with risk of thrombosis and myocardial infarction. This estimated response is comparable to the average of detailed anti-Neu5Gc IgG profile analyzed by a sialoglycan microarray. Both assays revealed an elevated response in acute KD patients with normal coronaries compared to patients with aneurysm or dilated coronaries. Implications of these findings are discussed.     

Prolonged stimuli alter the bacterial chemosensory clusters

The clustering of membrane‐bound receptors plays an essential role in various biological systems. A notable model system for studying this phenomenon is the bacterial chemosensory cluster that allows motile bacteria to navigate along chemical gradients in their environment. While the basic structure of these chemosensory clusters is becoming clear, their dynamic nature and operation are not yet understood. By measuring the fluorescence polarization of tagged receptor clusters in live Escherichia coli cells, we provide evidence for stimulus‐induced dynamics in these sensory clusters. We find that when a stimulus is applied, the packing of the receptors slowly decreases and that the process reverses when the stimulus is removed. Consistent with these physical changes we find that the effective cooperativity of the kinase response slowly evolves in the presence of a stimulus. Time‐lapse fluorescence imaging indicates that, despite these changes, the receptor clusters do not generally dissociate upon ligand binding. These data reveal stimulus‐dependent plasticity in chemoreceptor clusters.     

Proteasome inhibitors sensitize glioma cells and glioma stem cells to TRAIL-induced apoptosis by PKCε-dependent downregulation of AKT and XIAP expressions

In this study we examined the effects of proteasome inhibitors on cell apoptosis in TRAIL-resistant glioma cells and glioma stem cells (GSCs). Treatment with proteasome inhibitors and TRAIL induced apoptosis in all the resistant glioma cells and GSCs, but not in astrocytes and neural progenitor cells. Since PKCε has been implicated in the resistance of glioma cells to TRAIL, we examined its role in TRAIL and proteasome inhibitor-induced apoptosis. We found that TRAIL did not induce significant changes in the expression of PKCε, whereas a partial decrease in PKCε expression was obtained by proteasome inhibitors. A combined treatment of TRAIL and proteasome inhibitors induced accumulation of the catalytic fragment of PKCε and significantly and selectively decreased its protein and mRNA levels in the cancer but not in normal cells. Overexpression of PKCε partially inhibited the apoptotic effect of the proteasome inhibitors and TRAIL, and the caspase-resistant PKCεD383A mutant exerted a stronger inhibitory effect. Silencing of PKCε induced cell apoptosis in both glioma cells and GSCs, further supporting its role in cell survival. TRAIL and the proteasome inhibitors decreased the expression of AKT and XIAP in a PKCε-dependent manner and overexpression of these proteins abolished the apoptotic effect of this treatment. Moreover, silencing of XIAP sensitized glioma cells to TRAIL. Our results indicate that proteasome inhibitors sensitize glioma cells and GSCs to TRAIL by decreasing the expression of PKCε, AKT and XIAP. Combining proteasome inhibitors with TRAIL may be useful therapeutically in the treatment of gliomas and the eradication of GSCs.     

Intramolecular azo-bridge as a cystine disulfide bond surrogate: Somatostatin-14 and brain natriuretic peptide (BNP) analogs

Cystine disulfide bond is a common feature in numerous biologically active peptides and proteins and accordingly its replacement by various surrogates presents a potential route to obtain analogs with improved pharmacokinetic characteristics. The purpose of the present study was to assess whether an azo-bridge can serve as such a surrogate. In view of the marked clinical significance of somatostatin and the brain natriuretic peptide (BNP) we choose these peptides as a model. Three cyclic-azo somatostatin analogs and three cyclic-azo BNP analogs were effectively prepared in solution through azo bond formation between p-amino phenylalanine and His or Tyr residues that were positioned in the peptide sequences in place of the native Cys residues. The peptides binding affinities to the sst? and ANP-receptor (NPR-A) expressed on rat acinar pancreating carcinoma AR4-2J cell membranes and HeLa cells, respectively, were examined. The somatostatin analogs displayed good to moderate affinities to the rat sst? in the nM range with best results obtained with peptide 2, that is, IC?? = 8.1 nM. Molecular dynamics simulations on these peptides suggests on a correlation between the observed binding potencies and the degree of conformational space overlapping with that of somatostatin. The BNP analogs exhibited binding affinities to the NPR-A in the nM range with best results obtained with BNP-1, that is, IC?? = 60 nM.     

The Popdc gene family in the rat: molecular cloning, characterization and expression analysis in the heart and cultured cardiomyocytes

Three Popeye domain-containing (Popdc 1-3) family-members are known in vertebrates. Their exact function is as yet unknown although involvement in cell adhesion has been suggested. We report herein sequencing of the rat Popdc 1-3 cDNAs that show high homology to other vertebrate orthologs and are expressed primarily in the heart and skeletal muscles. Popdc2 splice variants were identified, with Popdc2C showing a distinctive age-dependent decline. In isolated cardiomyocytes, Popdc genes were negatively regulated by serum, an effect that was reversed by EGFR-kinase inhibition, suggesting an EGFR-dependent modulation of Popdc gene expression.     

Synergistic effects of abiotic stresses in plants: a case study of nitrogen limitation and saturating light intensity in Arabidopsis thaliana

Under natural conditions, plants are regularly exposed to combinations of stress factors. A common example is the conjunction between nitrogen (N) deficiency and excess light. The combined effect of stress factors is often ignored in studies using controlled conditions, possibly resulting in misleading conclusions. To address this issue, the present study examined the physiological behavior of Arabidopsis thaliana under the effect of varying nitrogen levels and light intensities. The joint influence of low N and excess light had an adverse effect on plant growth, chlorophyll and anthocyanin concentrations, photochemical capacity and the abundance of proteins involved in carbon assimilation and antioxidative metabolism. In contrast, no adverse physiological responses were observed for plants under either nitrogen limitation or high light (HL) intensity conditions (i.e. single stress). The underlying mechanisms for the increased growth in conditions of HL and sufficient nitrogen were a combination of chlorophyll accumulation and an increased number of proteins involved in C3 carbon assimilation, amino acids biosynthesis and chloroplast development. In contrast, combined stress conditions shifts plants from growth to survival by displaying anthocyanin accumulation and an increased number of proteins involved in catabolism of lipids and amino acids as energy substrates. Ultimately switching plants development from growth to survival. Our results suggest that an assessment of the physiological response to the combined effect of multiple stresses cannot be directly extrapolated from the physiological response to a single stress. Specifically, the synergistic interaction between N deficiency and saturating light in Arabidopsis plants could not have been modeled via only one of the stress factors.