Methylation is the initial reaction in anaerobic naphthalene degradation by a sulfate-reducing enrichment culture

The sulfate-reducing culture N47 can utilize naphthalene or 2-methylnaphthalene as the sole carbon source and electron donor. Here we show that the initial reaction in the naphthalene degradation pathway is a methylation to 2-methylnaphthalene which then undergoes the subsequent oxidation to the central metabolite 2-naphthoic acid, ring reduction and cleavage. Specific metabolites occurring exclusively during anaerobic degradation of 2-methylnaphthalene were detected during growth on naphthalene, i.e. naphthyl-2-methyl-succinate and naphthyl-2-methylene-succinate. Additionally, all three enzymes involved in anaerobic degradation of 2-methylnaphthalene to 2-naphthoic acid that could be measured in vitro so far, i.e. naphthyl-2-methyl-succinate synthase, succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase and naphthyl-2-methyl-succinyl-CoA dehydrogenase were also detected in naphthalene-grown cells with similar activities. Induction experiments were performed to study the growth behaviour of the cell when transferred from naphthalene to 2-methylnaphthalene or vice versa. When the cells were transferred from naphthalene to 2-methylnaphthalene they grew immediately, indicating that no new enzymes had to be induced. On the contrary, the transfer of cells from 2-methylnaphthalene to naphthalene caused a lag-phase of almost 100 days demonstrating that an additional catabolic enzyme has to be activated in this case. We propose the methylation as a novel general mechanism of activation reactions in anaerobic degradation of unsubstituted aromatic hydrocarbons.     

Score-based prediction of genomic islands in prokaryotic genomes using hidden Markov models

Background  

Horizontal gene transfer (HGT) is considered a strong evolutionary force shaping the content of microbial genomes in a substantial manner. It is the difference in speed enabling the rapid adaptation to changing environmental demands that distinguishes HGT from gene genesis, duplications or mutations. For a precise characterization, algorithms are needed that identify transfer events with high reliability. Frequently, the transferred pieces of DNA have a considerable length, comprise several genes and are called genomic islands (GIs) or more specifically pathogenicity or symbiotic islands.     

An integrated view of gene expression and solute profiles of Arabidopsis tumors: a genome-wide approach

Transformation of plant cells with T-DNA of virulent agrobacteria is one of the most extreme triggers of developmental changes in higher plants. For rapid growth and development of resulting tumors, specific changes in the gene expression profile and metabolic adaptations are required. Increased transport and metabolic fluxes are critical preconditions for growth and tumor development. A functional genomics approach, using the Affymetrix whole genome microarray (approximately 22,800 genes), was applied to measure changes in gene expression. The solute pattern of Arabidopsis thaliana tumors and uninfected plant tissues was compared with the respective gene expression profile. Increased levels of anions, sugars, and amino acids were correlated with changes in the gene expression of specific enzymes and solute transporters. The expression profile of genes pivotal for energy metabolism, such as those involved in photosynthesis, mitochondrial electron transport, and fermentation, suggested that tumors produce C and N compounds heterotrophically and gain energy mainly anaerobically. Thus, understanding of gene-to-metabolite networks in plant tumors promotes the identification of mechanisms that control tumor development.     

A novel fusion protein system for the production of native human pepsinogen in the bacterial periplasm

Human pepsinogen is the secreted inactive precursor of pepsin. Under the acidic conditions present in the stomach it is autocatalytically cleaved into the active protease. Pepsinogen contains three consecutive disulfides, and was used here as a model protein to investigate the production of aspartic proteases in the Escherichia coli periplasm. Various N-terminal translocation signals were applied and several different expression vectors were tested. After fusion to pelB, dsbA or ompT signal peptides no recombinant product could be obtained in the periplasm using the T7 promoter. As a new approach, human pepsinogen was fused to E. coli ecotin (E. coli trypsin inhibitor), which is a periplasmic homodimeric protein of 142 amino acids per monomer containing one disulfide bridge. The fusion protein was expressed in pTrc99a. After induction, the ecotin-pepsinogen fusion protein was translocated into the periplasm and the ecotin signal peptide was cleaved. Upon acid treatment, the fusion protein was converted into pepsin, indicating that pepsinogen was produced in its native form. In shake flasks experiments, the amount of active fusion protein present in the periplasm was 100 microg per litre OD 1, corresponding to 70 microg pepsinogen. After large scale cultivation, the fusion protein was isolated from the periplasmic extract. It was purified to homogeneity with a yield of 20%. The purified protein was native. Acid-induced activation of the fusion protein proceeded very fast. As soon as pepsin was present, the ecotin part of the fusion protein was rapidly digested, followed by a further activation of pepsinogen.     

Skull ontogeny: developmental patterns of fishes conserved across major tetrapod clades

In vertebrates, the ontogeny of the bony skull forms a particularly complex part of embryonic development. Although this area used to be restricted to neontology, recent discoveries of fossil ontogenies provide an additional source of data. One of the most detailed ossification sequences is known from Permo-Carboniferous amphibians, the branchiosaurids. These temnospondyls form a near-perfect link between the piscine osteichthyans and the various clades of extant tetrapods, retaining a full complement of dermal bones in the skull. For the first time, the broader evolutionary significance of these event sequences is analyzed, focusing on the identification of sequence heterochronies. A set of 120 event pairs was analyzed by event pair cracking, which helped identify active movers. A cladistic analysis of the event pair data was also carried out, highlighting some shared patterns between widely divergent clades of tetrapods. The analyses revealed an unexpected degree of similarity between the widely divergent taxa. Most interesting is the apparently modular composition of the cranial sequence: five clusters of bones were discovered in each of which the elements form in the same time window: (1) jaw bones, (2) marginal palatal elements, (3) circumorbital bones, (4) skull roof elements, and (5) neurocranial ossifications. In the studied taxa, these "modules" have in most cases been shifted fore and back on the trajectory relative to the Amia sequence, but did not disintegrate. Such "modules" might indicate a high degree of evolutionary limitation (constraint).     

Global down-regulation of gene expression in the brain using RNA interference, with emphasis on monoamine transporters and GPCRs: implications for target characterization in psychiatric and neurological disorders

RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.     

Identification of novel Kv1.3 blockers using a fluorescent cell-based ion channel assay

A functional cell-based assay was developed using a generic proprietary assay protocol, based on a membrane-potential sensitive dye, for the identification of small-molecule antagonists against the Kv1.3 potassium ion channel. A high-throughput screen (HTS) was subsequently performed with 20,000 compounds from the Evotec library, preselected using known small molecule antagonists for both sodium and potassium ion channels. Following data analysis, the hit rate was measured at 1.72%, and subsequent dose-response analysis of selected hits showed a high hit confirmation rate yielding approximately 50 compounds with an apparent IC50 value lower than 10 microM. Subsequent electrophysiological characterization of selected hits confirmed the initial activity and potency of the identified hits on the Kv1.3 target and also selectivity toward Kv1.3 through measurements on HERG as well as Kv1.3-expressing cell lines. Follow-up structure-activity relationship analysis revealed a variety of different clusters distributed throughout the library as well as several singlicates. In comparison to known Kv1.3 blockers, new chemical entities and scaffolds showing potency and selectivity against the Kv1.3 ion channel were detected. In addition, a screening strategy for ion channel drug discovery HTS, medicinal chemistry, and electrophysiology is presented.     

Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16

The H(2)-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H(2) and CO(2) as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism's ability to produce and store large amounts of poly[R-(-)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility.     

A cost-effective solution to reduce dead volume of a standard dispenser system by a factor of 5

A key trend in high-throughput screening is assay miniaturization to control reagent costs and increase throughput. For this purpose, liquid-handling devices are used that transfer nano-to low-microliter volumes into all currently used microtiter well plates. One drawback of many available dispenser and pipetting systems are high dead volumes. Therefore, the authors were looking for an easy and simple solution to modify their standard liquid-handling device, PerkinElmer's FlexDrop Precision IV, allowing for a dead volume reduction to receive maximum benefit from miniaturized assay formats. Internal reservoirs were developed and constructed by Schering's Technical Development Laboratory (TDL), which are directly connected to the dispenser banks of FlexDrop without tubing. Using these newly built reservoirs, the dead volume was decreased by a factor of 5 in comparison to the manufacturer's reservoirs without compromising liquid-handling parameters such as accuracy and precision. The modified system displayed a high robustness and reliability under routine high-throughput screening conditions.     

A lock-and-key model for protein-protein interactions

MOTIVATION: Protein-protein interaction networks are one of the major post-genomic data sources available to molecular biologists. They provide a comprehensive view of the global interaction structure of an organism's proteome, as well as detailed information on specific interactions. Here we suggest a physical model of protein interactions that can be used to extract additional information at an intermediate level: It enables us to identify proteins which share biological interaction motifs, and also to identify potentially missing or spurious interactions. RESULTS: Our new graph model explains observed interactions between proteins by an underlying interaction of complementary binding domains (lock-and-key model). This leads to a novel graph-theoretical algorithm to identify bipartite subgraphs within protein-protein interaction networks where the underlying data are taken from yeast two-hybrid experimental results. By testing on synthetic data, we demonstrate that under certain modelling assumptions, the algorithm will return correct domain information about each protein in the network. Tests on data from various model organisms show that the local and global patterns predicted by the model are indeed found in experimental data. Using functional and protein structure annotations, we show that bipartite subnetworks can be identified that correspond to biologically relevant interaction motifs. Some of these are novel and we discuss an example involving SH3 domains from the Saccharomyces cerevisiae interactome. AVAILABILITY: The algorithm (in Matlab format) is available (see http://www.maths.strath.ac.uk/~aas96106/lock_key.html).