16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales"

For simultaneous identification of members of the betaproteobacterial order "Rhodocyclales" in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the "Rhodocyclales." The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent "Rhodocyclales" diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed "Rhodocyclales"-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of "Rhodocyclales" populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.     

The biophysical and molecular basis of TRPV1 proton gating

The capsaicin receptor TRPV1, a member of the transient receptor potential family of non-selective cation channels is a polymodal nociceptor. Noxious thermal stimuli, protons, and the alkaloid irritant capsaicin open the channel. The mechanisms of heat and capsaicin activation have been linked to voltage-dependent gating in TRPV1. However, until now it was unclear whether proton activation or potentiation or both are linked to a similar voltage-dependent mechanism and which molecular determinants underlie the proton gating. Using the whole-cell patch-clamp technique, we show that protons activate and potentiate TRPV1 by shifting the voltage dependence of the activation curves towards more physiological membrane potentials. We further identified a key residue within the pore region of TRPV1, F660, to be critical for voltage-dependent proton activation and potentiation. We conclude that proton activation and potentiation of TRPV1 are both voltage dependent and that amino acid 660 is essential for proton-mediated gating of TRPV1.     

Prediction of effective genome size in metagenomic samples

We introduce a novel computational approach to predict effective genome size (EGS; a measure that includes multiple plasmid copies, inserted sequences, and associated phages and viruses) from short sequencing reads of environmental genomics (or metagenomics) projects. We observe considerable EGS differences between environments and link this with ecologic complexity as well as species composition (for instance, the presence of eukaryotes). For example, we estimate EGS in a complex, organism-dense farm soil sample at about 6.3 megabases (Mb) whereas that of the bacteria therein is only 4.7 Mb; for bacteria in a nutrient-poor, organism-sparse ocean surface water sample, EGS is as low as 1.6 Mb. The method also permits evaluation of completion status and assembly bias in single-genome sequencing projects.     

Co-expression of nicastrin and presenilin rescues a loss of function mutant of APH-1

gamma-Secretase is an intramembrane-cleaving aspartyl protease complex that mediates the final cleavage of beta-amyloid precursor protein to liberate the neurotoxic amyloid-beta peptide implicated in Alzheimer's disease. The four proteins presenilin (PS), nicastrin (NCT), APH-1, and PEN-2 are sufficient to reconstitute gamma-secretase activity in yeast. Although PS seems to contribute the catalytic core of the gamma-secretase complex, no distinct function could be attributed to the other components so far. In Caenorhabditis elegans, mutation of a glycine to an aspartic acid within a conserved GXXXG motif in the fourth transmembrane domain of APH-1 causes a loss of function phenotype. Surprisingly, we now found that the human homologue APH-1a carrying the equivalent mutation G122D is fully active in yeast co-expressing PS1, NCT, and PEN-2. To address this discrepancy, we expressed APH-1a G122D in HEK293 cells. As reported previously, overexpressed APH-1a G122D was not incorporated into the gamma-secretase complex. Separate overexpression of PS1, NCT, or PEN-2 together with APH-1a G122D allowed the formation of heterodimers lacking the other endogenous components. Only the combined overexpression of PS1 and NCT together with APH-1a G122D facilitated the formation of a fully active gamma-secretase complex. Under these conditions, APH-1a G122D supported the production of normal amounts of Abeta. We conclude that cooperative effects may stabilize a trim-eric complex of APH-1a G122D together with PS1 and NCT. Upon successful complex assembly, the GXXXG motif becomes dispensable for gamma-secretase activity.     

Composition, and antimicrobial and remarkable antiprotozoal activities of the essential oil of rhizomes of Aframomum sceptrum K. Schum. (Zingiberaceae)

The essential oil from the rhizomes of Aframomum sceptrum (Zingiberaceae) was analyzed by GC/MS, and its major constituents were found to be β‐pinene (12.7%), caryophyllene oxide (10.0%), and cyperene (6.0%). The oil was also evaluated for antimicrobial activities, in comparison with β‐pinene, caryophyllene oxide, and the leaf essential oil of Melaleuca alternifolia (Myrtaceae). The A. sceptrum essential oil exhibited bacteriostatic activity against the Gram‐positive bacteria Bacillus subtilis, Staphylococcus epidermidis, and S. aureus, but not against Gram‐negative bacteria. Moreover, it showed mild fungicidal activity against Candida albicans and Aspergillus fumigates, and remarkable antiprotozoal activity against Trypanosoma brucei brucei (MLC of 1.51 μl/ml) and Trichomonas vaginalis (IC₅₀ of 0.12±0.02 and MLC of 1.72 μl/ml).     

Human periosteum-derived progenitor cells express distinct chemokine receptors and migrate upon stimulation with CCL2, CCL25, CXCL8, CXCL12, and CXCL13

For bone repair, transplantation of periosteal progenitor cells (PCs), which had been amplified within supportive scaffolds, is applied clinically. More innovative bone tissue engineering approaches focus on the in situ recruitment of stem and progenitor cells to defective sites and their subsequent use for guided tissue repair. Chemokines are known to induce the directed migration of bone marrow CD34(-) mesenchymal stem cells (MSCs). The aim of our study was to determine the chemokine receptor expression profile of human CD34(-) PCs and to demonstrate that these cells migrate upon stimulation with selected chemokines. PCs were isolated from periosteum of the mastoid bone and displayed a homogenous cell population presenting an MSC-related cell-surface antigen profile (ALCAM(+), SH2(+), SH3(+), CD14(-), CD34(-), CD44(+), CD45(-), CD90(+)). The expression profile of chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that PCs express receptors of all four chemokine subfamilies CC, CXC, CX(3)C, and C. Migration of PCs and a dose-dependent migratory effect of the chemokines CCL2 (MCP1), CCL25 (TECK), CXCL8 (IL8), CXCL12 (SDF1alpha), and CXCL13 (BCA1), but not CCL22 (MDC) were demonstrated using a 96-multiwell chemotaxis assay. In conclusion, for the first time, here we report that human PCs express chemokine receptors, present their profile, and demonstrate a dose-dependent migratory effect of distinct chemokines on these cells. These results are promising towards in situ bone repair therapies based on guiding PCs to bone defects, and encourage further in vivo studies.     

Tyramine and octopamine: antagonistic modulators of behavior and metabolism

The phenolamines tyramine and octopamine are decarboxylation products of the amino acid tyrosine. Although tyramine is the biological precursor of octopamine, both compounds are independent neurotransmitters, acting through various G-protein coupled receptors. Especially, octopamine modulates a plethora of behaviors, peripheral and sense organs. Both compounds are believed to be homologues of their vertebrate counterparts adrenaline and noradrenaline. They modulate behaviors and organs in a coordinated way, which allows the insects to respond to external stimuli with a fine tuned adequate response. As these two phenolamines are the only biogenic amines whose physiological significance is restricted to invertebrates, the attention of pharmacologists was focused on the corresponding receptors, which are still believed to represent promising targets for new insecticides. Recent progress made on all levels of octopamine/tyramine research enabled us to better understand the molecular events underlying the control of complex behaviors.     

Solubilization of Sodium Channel from Human Brain

[3H]Tetrodotoxin binds to a single class of receptor sites in homogenates of human brain with a KD of 9.1 nM at 0 degree C and a maximal binding capacity of 5.9 pmol/mg of protein. This tetrodotoxin receptor has been solubilized, and several parameters influencing the efficiency of this critical step have been studied. Treatment of brain membranes with 2% (wt/vol) Nonidet P-40 solubilizes up to 38% of the tetrodotoxin receptor sites. The duration of this solubilization step must not exceed 15 min at an optimal pH of 6.8. The binding activity is most stable when exogenous phosphatidylcholine is added to the soluble receptor with a phosphatidylcholine/detergent ratio of 1:5.     

Evolved distal tail carbohydrate binding modules of Lactobacillus phage J‐1: a novel type of anti‐receptor widespread among lactic acid bacteria phages

Bacteriophage replication requires specific host‐recognition. Some siphophages harbour a large complex, the baseplate, at the tip of their non‐contractile tail. This baseplate holds receptor binding proteins (RBPs) that can recognize the host cell‐wall polysaccharide (CWPS) and specifically attach the phage to its host. While most phages possess a dedicated RBP, the phage J‐1 that infects Lactobacillus casei seemed to lack one. It has been shown that the phage J‐1 distal tail protein (Dit) plays a role in host recognition and that its sequence comprises two inserted modules compared with ‘classical’ Dits. The first insertion is similar to carbohydrate‐binding modules (CBMs), whereas the second insertion remains undocumented. Here, we determined the structure of the second insertion and found it also similar to several CBMs. Expressed insertion CBM2, but not CBM1, binds to L. casei cells and neutralize phage attachment to the bacterial cell wall and the isolated and purified CWPS of L. casei BL23 prevents CBM2 attachment to the host. Electron microscopy single particle reconstruction of the J‐1 virion baseplate revealed that CBM2 is projected at the periphery of Dit to optimally bind the CWPS receptor. Taken together, these results identify J‐1 evolved Dit as the phage RBP.