PASSAGE OF 5-HYDROXYTRYPTAMINE THROUGH THE BLOOD-BRAIN BARRIER, ITS METABOLISM IN THE BRAIN AND ELIMINATION OF 5-HYDROXYINDO-LEACETIC ACID FROM THE BRAIN TISSUE

Abstract— 5-HT was injected intravenously in rats (10 mg/kg) and a marked increase in brain 5-HT and 5-HIAA was observed. For the first 10 min after injection the penetration of 5-HT into the brain and formation of 5-HIAA is evident. After 10 min degradation of exogenous 5-HT and elimination of 5-HIAA are prominent. Metabolism of exogenous 5-HT in the brain is very fast (half-life between 5 and 10 min; completely metabolized in approximately 80 min). The importance of these results in explaining the permeability of blood-brain barrier to 5-HT is discussed. Experiments on brain slices show that 5-HT is more readily metabolized in brain tissue than eliminated into incubation medium. In contrast, 5-HIAA very easily leaves brain tissue.     

Phyletic Profiling with Cliques of Orthologs Is Enhanced by Signatures of Paralogy Relationships

New microbial genomes are sequenced at a high pace, allowing insight into the genetics of not only cultured microbes, but a wide range of metagenomic collections such as the human microbiome. To understand the deluge of genomic data we face, computational approaches for gene functional annotation are invaluable. We introduce a novel model for computational annotation that refines two established concepts: annotation based on homology and annotation based on phyletic profiling. The phyletic profiling-based model that includes both inferred orthologs and paralogs—homologs separated by a speciation and a duplication event, respectively—provides more annotations at the same average Precision than the model that includes only inferred orthologs. For experimental validation, we selected 38 poorly annotated Escherichia coli genes for which the model assigned one of three GO terms with high confidence: involvement in DNA repair, protein translation, or cell wall synthesis. Results of antibiotic stress survival assays on E. coli knockout mutants showed high agreement with our model''s estimates of accuracy: out of 38 predictions obtained at the reported Precision of 60%, we confirmed 25 predictions, indicating that our confidence estimates can be used to make informed decisions on experimental validation. Our work will contribute to making experimental validation of computational predictions more approachable, both in cost and time. Our predictions for 998 prokaryotic genomes include ∼400000 specific annotations with the estimated Precision of 90%, ∼19000 of which are highly specific—e.g. “penicillin binding,” “tRNA aminoacylation for protein translation,” or “pathogenesis”—and are freely available at http://gorbi.irb.hr/.     

INCA: synonymous codon usage analysis and clustering by means of self-organizing map

SUMMARY: INteractive Codon usage Analysis (INCA) provides an array of features useful in analysis of synonymous codon usage in whole genomes. In addition to computing codon frequencies and several usage indices, such as 'codon bias', effective Nc and CAI, the primary strength of INCA has numerous options for the interactive graphical display of calculated values, thus allowing visual detection of various trends in codon usage. Finally, INCA includes a specific unsupervised neural network algorithm, the self-organizing map, used for gene clustering according to the preferred utilization of codons. AVAILABILITY: INCA is available for the Win32 platform and is free of charge for academic use. For details, visit the web page http://www.bioinfo-hr.org/inca or contact the author directly. SUPPLEMENTARY INFORMATION: Software is accompanied with a user manual and a short tutorial.     

Translational Selection Is Ubiquitous in Prokaryotes

Codon usage bias in prokaryotic genomes is largely a consequence of background substitution patterns in DNA, but highly expressed genes may show a preference towards codons that enable more efficient and/or accurate translation. We introduce a novel approach based on supervised machine learning that detects effects of translational selection on genes, while controlling for local variation in nucleotide substitution patterns represented as sequence composition of intergenic DNA. A cornerstone of our method is a Random Forest classifier that outperformed previous distance measure-based approaches, such as the codon adaptation index, in the task of discerning the (highly expressed) ribosomal protein genes by their codon frequencies. Unlike previous reports, we show evidence that translational selection in prokaryotes is practically universal: in 460 of 461 examined microbial genomes, we find that a subset of genes shows a higher codon usage similarity to the ribosomal proteins than would be expected from the local sequence composition. These genes constitute a substantial part of the genome—between 5% and 33%, depending on genome size—while also exhibiting higher experimentally measured mRNA abundances and tending toward codons that match tRNA anticodons by canonical base pairing. Certain gene functional categories are generally enriched with, or depleted of codon-optimized genes, the trends of enrichment/depletion being conserved between Archaea and Bacteria. Prominent exceptions from these trends might indicate genes with alternative physiological roles; we speculate on specific examples related to detoxication of oxygen radicals and ammonia and to possible misannotations of asparaginyl–tRNA synthetases. Since the presence of codon optimizations on genes is a valid proxy for expression levels in fully sequenced genomes, we provide an example of an “adaptome” by highlighting gene functions with expression levels elevated specifically in thermophilic Bacteria and Archaea.     

Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin

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Highlights? The fungal secondary metabolite Cladosporin inhibits liver- and blood-stage malaria parasites ? Cladosporin specifically targets lysyl-tRNA synthetase (Krs1) ? Cladosporin is >100-fold more potent against parasite Krs1 relative to the human enzyme ? Two amino acids in the Krs1 ATP-binding pocket confer species-selective inhibition     

Identification of human kinases involved in hepatitis C virus replication by small interference RNA library screening

The propagation of the hepatitis C virus (HCV) is a complex process that requires both host and viral proteins. To facilitate identification of host cell factors that are required for HCV replication, we screened a panel of small interference RNAs that preferentially target human protein kinases using an HCV replicon expressing the firefly luciferase gene as a genetic reporter. Small interference RNAs specific for three human kinases, Csk, Jak1, and Vrk1, were identified that reproducibly reduce viral RNA and viral protein levels in HCV replicon-bearing cells. Treatment of replicon cells with a small molecule inhibitor of Csk also resulted in a significant reduction in HCV RNA and proteins, further supporting a role for Csk in HCV replication. The effects of siRNAs targeting eight kinases known to be negatively regulated by Csk were then examined; knock down of one of these kinases, Fyn, resulted in up-regulation of the HCV replicon, suggesting that Csk mediates its effect on HCV replication through Fyn. This conclusion was further corroborated by demonstration that replicon cells treated with Csk inhibitor contained lower levels of the phosphorylated form of Fyn than control cells.     

Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc₁) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, Q_N and Q_P. The L197F mutation is located in the Q_N site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another Q_N site inhibitor, but not to strobilurin or myxothiazol, which target the Q_P site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC₅₀) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.