Predicting Kinase Activity in Angiotensin Receptor Phosphoproteomes Based on Sequence-Motifs and Interactions

Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT_1aR) is one of the most studied 7TMRs with respect to selective activation of the β-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT_1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT_1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.     

Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability

Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using material from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within genera containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. A representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation.     

Analysis of the genetic determinant for production of the peptide antibiotic nisin

The structural gene for the precursor of the peptide antibiotic nisin was isolated and characterized. As with other lanthionine-containing antibiotics, nisin is synthesized as a pre-propeptide which undergoes post-translational modification to generate the mature antibiotic. The sequence data obtained agreed with those of precursor nisin genes isolated by other workers from different Lactococcus lactis strains. Analysis of regions flanking the precursor nisin gene revealed the presence of a downstream open reading frame that may be involved in maturation of the precursor molecule. Nucleotide sequences characteristic of an IS element were located upstream of the nisin determinant. This element, termed IS904, is present in multiple copies in the genome of L. lactis. The nisin determinant of L. lactis is a component of a large transmissible gene block that also encodes nisin resistance and sucrose-metabolizing genes. Gene probe experiments indicated that the nisin/sucrose gene block was located in the chromosome. Furthermore, the copy of IS904 identified adjacent to the precursor nisin gene lies at, or very close to, one end of this transmissible DNA segment and may play a role in mediating its transfer between strains.     

Direct measurement of the force between two lipid bilayers and observation of their fusion

The force between two phosphatidylcholine bilayers is measured as a function of their separation, showing a strong hydration repulsion at short range, as previously reported by LeNeveu et al. (LeNeveu, D.M., Rand, R.P., Parsegian, V.A. and Gingell, D. (1977) (Biophys. J. 18, 209–230). The experimental technique also allows direct observation of the molecular process by which two bilayers fuse into one.     

Variability in the role of the gasbladder in fish audition

The teleost gasbladder is believed to aid in fish audition by transferring pressure components of incoming sound to the inner ears. This idea is primarily based on both anatomical observations of the mechanical connection between the gasbladder and the ear, followed by physiological experiments by various researchers. The gasbladder movement has been modeled mathematically as a pulsating bubble. This study is extending the previous work on fish with a physical coupling of the gasbladder and ear by investigating hearing in two species (the blue gourami Trichogaster trichopterus, and the oyster toadfish Opsanus tau) without a mechanical linkage. An otophysan specialist (the goldfish Carassius auratus) with mechanical coupling, is used as the control. Audiograms were obtained with acoustically evoked potentials (e.g., auditory brainstem response) from intact fish and from the same individuals with their gasbladders deflated. In blue gourami and oyster toadfish, removal of gas did not significantly change thresholds, and evoked potentials had similar waveforms. In goldfish thresholds increased by 33–55 dB (frequency dependent) after deflation, and major changes in evoked potentials were observed. These results suggest that the gasbladder may not serve an auditory enhancement function in teleost fishes that lack mechanical coupling between the gasbladder and the inner ear. Accepted: 28 February 2000