Identification of cisapride as new inhibitor of putrescine uptake in Trypanosoma cruzi by combined ligand- and structure-based virtual screening.

Nowadays, the pharmacological therapy for the treatment of Chagas disease is based on two old drugs, benznidazole and nifurtimox, which have restricted efficacy against the chronic phase of the illness. To overcome the lack of efficacy of the traditional drugs (and their considerable toxicity), new molecular targets have been studied as starting points to the discovery of new antichagasic compounds. Among them, polyamine transporter TcPAT12 (also known as TcPOT1.1) represents an interesting macromolecule, since polyamines are essential for Trypanosoma cruzi, the parasite that causes the illness, but it cannot synthesize them de novo. In this investigation we report the results of a combined ligand- and structure-based virtual screening for the discovery of new inhibitors of TcPAT12. Initially we filtered out ZINC and Drugbank databases with similarity and QSAR models and then we submitted the candidates to a validated docking based screening. Four structures were selected and tested in T. cruzi epimastigotes proliferation and two of them, Cisapride and [2-(cyclopentyloxy)phenyl]methanamine showed inhibitory effects. Additionally, we performed transport assays which demonstrated that Cisapride interferes with putrescine uptake in a specific mode.

Can root electrical capacitance be used to predict root mass in soil?

BACKGROUND: Electrical capacitance, measured between an electrode inserted at the base of a plant and an electrode in the rooting substrate, is often linearly correlated with root mass. Electrical capacitance has often been used as an assay for root mass, and is conventionally interpreted using an electrical model in which roots behave as cylindrical capacitors wired in parallel. Recent experiments in hydroponics show that this interpretation is incorrect and a new model has been proposed. Here, the new model is tested in solid substrates. METHODS: The capacitances of compost and soil were determined as a function of water content, and the capacitances of cereal plants growing in sand or potting compost in the glasshouse, or in the field, were measured under contrasting irrigation regimes. KEY RESULTS: Capacitances of compost and soil increased with increasing water content. At water contents approaching field capacity, compost and soil had capacitances at least an order of magnitude greater than those of plant tissues. For plants growing in solid substrates, wetting the substrate locally around the stem base was both necessary and sufficient to record maximum capacitance, which was correlated with stem cross-sectional area: capacitance of excised stem tissue equalled that of the plant in wet soil. Capacitance measured between two electrodes could be modelled as an electrical circuit in which component capacitors (plant tissue or rooting substrate) are wired in series. CONCLUSIONS: The results were consistent with the new physical interpretation of plant capacitance. Substrate capacitance and plant capacitance combine according to standard physical laws. For plants growing in wet substrate, the capacitance measured is largely determined by the tissue between the surface of the substrate and the electrode attached to the plant. Whilst the measured capacitance can, in some circumstances, be correlated with root mass, it is not a direct assay of root mass.

Alternative splicing of U12-dependent introns in vivo responds to purine-rich enhancers.

Alternative splicing increases the coding capacity of genes through the production of multiple protein isoforms by the conditional use of splice sites and exons. Many alternative splice sites are regulated by the presence of purine-rich splicing enhancer elements (ESEs) located in the downstream exon. Although the role of ESEs in alternative splicing of the major class U2-dependent introns is well established, no alternatively spliced minor class U12-dependent introns have so far been described. Although in vitro studies have shown that ESEs can stimulate splicing of individual U12-dependent introns, there is no direct evidence that the U12-dependent splicing system can respond to ESEs in vivo. To investigate the ability of U12-dependent introns to use alternative splice sites and to respond to ESEs in an in vivo context, we have constructed two sets of artificial minigenes with alternative splicing pathways and evaluated the effects of ESEs on their alternative splicing patterns. In minigenes with alternative U12-dependent 3' splice sites, a purine-rich ESE promotes splicing to the immediately upstream 3' splice site. As a control, a mutant ESE has no stimulatory effect. In minigene constructs with two adjacent U12-dependent introns, the predominant in vivo splicing pattern results in the skipping of the internal exon. Insertion of a purine-rich ESE into the internal exon promotes the inclusion of the internal exon. These results show that U12-dependent introns can participate in alternative splicing pathways and that U12-dependent splice sites can respond to enhancer elements in vivo.

Role of the 3' splice site in U12-dependent intron splicing.

U12-dependent introns containing alterations of the 3' splice site AC dinucleotide or alterations in the spacing between the branch site and the 3' splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5' splice site AU dinucleotide, any nucleotide could serve as the 3'-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3' splice site function. A branch site-to-3' splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3' splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3' splice site but that formation of the prespliceosomal complex A requires an active 3' splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3' splice site.

Terminal intron dinucleotide sequences do not distinguish between U2- and U12-dependent introns.

Two types of eukaryotic nuclear introns are known: the common U2-dependent class with /GU and AG/ terminal intron dinucleotides, and the rare U12-dependent class with /AU and AC/ termini. Here we show that the U12-dependent splicing system can splice introns with /GU and AG/ termini and that such introns occur naturally. Further, U2-dependent introns with /AU and AC/termini also occur naturally and are evolutionarily conserved. Thus, the sequence of the terminal dinucleotides does not determine which spliceosomal system removes an intron. Rather, the four classes of introns described here can be sorted into two mechanistic classes (U2- or U12-dependent) by inspection of the complete set of conserved splice site sequences.

Mutation of the conserved first nucleotide of a group II intron from yeast mitochondrial DNA reduces the rate but allows accurate splicing.

Group II introns have a phylogenetically conserved 5'-terminal pentanucleotide, -G1U2G3C4G5-, that resembles the conserved 5' end sequence of nuclear pre-mRNA introns. No functional interaction or catalytic role for the conserved G1 position has been proposed, although a tertiary structure involving -G3C4- has been implicated in splicing in vitro. We have analyzed splicing phenotypes both in vitro and in vivo for all three point mutants affecting guanosine at position 1 (G1) of intron 5 gamma from the COXI gene of yeast mitochondrial DNA. While all of these G1N substitutions slow splicing in vitro, G1C is clearly the most defective. All three mutant transcripts splice as accurately as the wild-type transcript, although the yield of lariat intron is reduced. The branched trinucleotide core includes the mutated position 1 nucleotide linked to the canonical branchpoint adenosine. The mutant lariats vary significantly in their susceptibility to the debranching activity from human cells. After wild-type, G1A was most sensitive, G1U was somewhat resistant, while G1C was highly resistant to debranching. These mutant lariats had normal ribozyme activity for promoting spliced exon reopening. The three mutant introns were transformed into otherwise normal yeast mitochondrial DNA. These mutants grow on nonfermentable carbon sources and splce aI5 gamma to yield excised intron lariat and mRNA. Nonetheless, each mutant splices with reduced efficiency, roughly parallel to their in vitro activity. In vivo, all three mutants accumulate both the pre-mRNA retaining intron 5 gamma and the lariat splicing intermediate containing intron and 3' exon. Clearly, this primary sequence element, shared with nuclear pre-mRNA introns, has a very different functional significance in group II splicing.

Group II intron domain 5 facilitates a trans-splicing reaction.

A self-splicing group II intron of yeast mitochondrial DNA (aI5g) was divided within intron domain 4 to yield two RNAs that trans-spliced in vitro with associated trans-branching of excised intron fragments. Reformation of the domain 4 secondary structure was not necessary for the trans reaction, since domain 4 sequences were shown to be dispensable. Instead, the trans reaction depended on a previously unpredicted interaction between intron domain 5, the most highly conserved region of group II introns, and another region of the RNA. Domain 5 was shown to be essential for cleavage at the 5' splice site. It stimulated that cleavage when supplied as a trans-acting RNA containing only 42 nucleotides of intron sequence. The relevance of our findings to in vivo trans-splicing mechanisms is discussed.

Group II intron self-splicing. Alternative reaction conditions yield novel products.

Reaction parameters were modified to enhance the in vitro reaction rate and to reveal partial and novel reactions of the group II intron 5g of the mitochondrial gene from Saccharomyces cerevisiae encoding cytochrome c oxidase subunit I. One alteration yields separate 5'- and 3'-exons plus linear excised intron as the main products. A linear reaction intermediate, containing intron and 3'-exon, and products resulting from cleavages at two unexpected sites were identified. Spliced exon "reopening," a novel reaction between excised intron and spliced exons, appears responsible for separate 5'- and 3'-exon products.