State-of-the-art in phosphoproteomics

Presently, phosphorylation of proteins is the most studied and best understood PTM. However, the analysis of phosphoproteins and phosphopeptides is still one of the most challenging tasks in contemporary proteome research. Since not every phosphoprotein is accessible by a certain method and identification of the phosphorylated amino acid residue is required in the majority of cases, various strategies for the detection and localization of phosphorylations have been developed. Identification and localization of protein phosphorylations is mostly done by MS nowadays but phosphoproteins and -peptides are often suppressed in comparison to the unphosphorylated species if measured in complex mixtures. Thus, the isolation of pure phosphopeptide samples is a main task. This review gives an overview over the most frequently used methods in isolation and detection of phosphoproteins and -peptides such as specific enrichment or separation strategies as well as the localization of the phosphorylated residues by various mass spectrometric techniques.     

Linkage of the DNA-segment D7S13 (pB79a) with the cystic fibrosis locus

Summary A map distance of 2.9 cM between D7S13 (pB79a) and the cystic fibrosis (CF) locus was obtained from the analysis of 13 informative families with a history of CF. This result is based solely on the HindIII restriction fragment length polymorphism (HindIII-RFLP) at D7S13, since the interpretation of the Msp1-RFLP at this locus was found to be unreliable.     

Culture-independent characterization of the digestive-tract microbiota of the medicinal leech reveals a tripartite symbiosis

Culture-based studies of the microbial community within the gut of the medicinal leech have typically been focused on various Aeromonas species, which were believed to be the sole symbiont of the leech digestive tract. In this study, analysis of 16S rRNA gene clone libraries confirmed the presence of Aeromonas veronii and revealed a second symbiont, clone PW3, a novel member of the Rikenellaceae, within the crop, a large compartment where ingested blood is stored prior to digestion. The diversity of the bacterial community in the leech intestinum was determined, and additional symbionts were detected, including members of the alpha-, gamma-, and delta-Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. The relative abundances of the clones suggested that A. veronii and the novel clone, PW3, also dominate the intestinum community, while other clones, representing transient organisms, were typically present in low numbers. The identities of these transients varied greatly between individual leeches. Neither time after feeding nor feeding on defibrinated blood caused a change in identity of the dominant members of the microbial communities. Terminal restriction fragment length polymorphism analysis was used to verify that the results from the clone libraries were representative of a larger data set. The presence of a two-member bacterial community in the crop provides a unique opportunity to investigate both symbiont-symbiont and symbiont-host interactions in a natural model of digestive-tract associations.     

Outgrowing olfactory axons contain the Reelin receptor VLDLR and navigate through the Reelin-rich cribriform mesenchyme

Chemosensory neurons in the olfactory epithelium (OE) project axonal processes to the olfactory bulb (OB) of the brain. During embryonic stages, on their trajectory to the OB, the outgrowing axons traverse the so-called cribriform mesenchyme, which is located between the OE and the OB. The molecular cues guiding these axons through the cribriform mesenchyme are largely unknown. To identify molecules influencing the axonal trajectory in the murine cribriform mesenchyme, we performed microarray analyses focusing on extracellular matrix (ECM) proteins present in this tissue. Thereby, the ECM protein Reelin turned out to be an interesting candidate. Reelin was found to be expressed by numerous cells in the cribriform mesenchyme during the embryonic stages when the first axons navigate from the OE to the OB. These cells were closely associated with olfactory axons and apparently lack glial and neuronal markers. In the mesenchyme underlying the OE, localization of the Reelin protein was not confined to the Reelin-expressing cells, but it was also observed to be widely distributed in the ECM—most prominently in regions traversed by olfactory axons. Importantly, these axons were found to be endowed with the Reelin receptor very-low-density lipoprotein receptor (VLDLR). Finally, Reelin expression was also detectable in neuronal cells of the OB, which are contacted by VLDLR-positive olfactory axons. In summary, the results of the present study suggest that a Reelin/VLDLR signaling pathway might contribute to the formation of olfactory projections to the OB and the establishment of initial contacts between the incoming axons and neurons in the OB. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Funding:  This work was supported by the Deutsche Forschungsgemeinschaft.