Topoisomerases inhibitory activities and DNA binding properties of 9-methoxycamptothecin from <i>Nothapodytes nimmoniana (J. Graham) Mabberly</i>

<p>We have reported previously that 9-methoxycamptothecin (MCPT) showed significant antitumor activity <i>in vitro</i>. Here, agarose gel electrophoresis experiments were performed to evaluate MCPT’s unwinding ability toward plasmid DNA and inhibitory activities against topoisomerases (Topo) I and II. Binding properties of MCPT to calf thymus DNA (CT-DNA) were evaluated by UV–vis, melting temperature, fluorescence, circular dichroism methodologies and molecular docking technique. Results showed that MCPT at 100 μM inhibited Topo I activity, but had no effect on Topo II. Studies on the binding properties indicated that minor groove binding was the most probable binding mode of MCPT to DNA. The abilities of MCPT to act as Topo I inhibitor and minor groove binding agent may be related to its strong antitumor activity.</p

Characterization of <i>Pythium</i> Transcriptome and Gene Expression Analysis at Different Stages of Fermentation

<div><p>Background</p><p>The <i>Pythium splendens</i> is a potentially useful organism for the synthesis of large amounts of eicosapentaenoic acid. Peak biomass and lipid accumulation do not occur at the same time and growth temperature has an effect on the fatty acid composition. Little is known about the pathway or the genes involved in growth, lipid synthesis or temperature resistance in <i>P. splendens</i>. Analysis of the transcriptome and expression profile data for <i>P.splendens</i>RBB-5 were used to extend genetic information for this strain and to contribute to a comprehensive understanding of the molecular mechanisms involved in specific biological processes.</p><p>Methodology/Principal Findings</p><p>This study used transcriptome assembly and gene expression analysis with short-read sequencing technology combined with a tag-based digital gene expression (DGE) system. Assembled sequences were annotated with gene descriptions, such as gene ontology (GO), clusters of orthologous group (COG) terms and KEGG orthology (KO) to generate 23,796 unigenes. In addition, we obtained a larger number of genes at different stages of fermentation (48, 100 and 148 h). The genes related to growth characteristics and lipid biosynthesis were analyzed in detail. Some genes associated with lipid and fatty acid biosynthesis were selected to confirm the digital gene expression (DGE) results by quantitative real-time PCR (qRT-PCR).</p><p>Conclusion/Significance</p><p>The transcriptome improves our genetic understanding of <i>P.splendens</i>RBB-5 greatly and makes a large number of gene sequences available for further study. Notably, the transcriptome and DGE profiling data of <i>P.splendens</i>RBB-5 provide a comprehensive insight into gene expression profiles at different stages of fermentation and lay the foundation for the study of optimizing lipid content and growth speed at the molecular level.</p></div

COG function classification of all unigenes from transcriptome of <i>P.splendens</i>.

<p>A, RNA processing and modification; B, Chromatin structure and dynamics; C, Energy production and conversion; D, Cell cycle control, cell division, chromosome partitioning; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism; G, Carbohydrate transport and metabolism; H, Coenzyme transport and metabolism; I, Lipid transport and metabolism; J, Translation, ribosomal structure and biogenesis; K, Transcription; L, Replication, recombination and repair; M, Cell wall/membrane/envelope biogenesis; N, Cell motility; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabolism; R, General function prediction only; S, Function unknown; T, Signal transduction mechanisms; U, Intracellular trafficking, secretion, and vesicular transport; V, Defense mechanisms; W, Extracellular structures; Y, Nuclear structure; Z, Cytoskeleton.</p

Unigenes length distribution of <i>P. splendens</i>

<p>Unigenes length distribution of <i>P. splendens</i></p

Validation of the gene expression profile by qRT-PCR.

<p>PDDA: phospho-2-dehydro-3-deoxyheptonate aldolase, ACOX: acyl-coenzyme an oxidase, ACO: aconitate hydratase, D12: Δ12 fatty acid desaturase, D9: Δ9 fatty acid desaturase, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, GPDH: Glycerol-3-phosphate dehydrogenase, GTP: phosphoenolpyruvate carboxykinase; *P<0.05, **P<0.01.</p

Contigs length distribution of <i>P. splendens</i>.

<p>Contigs length distribution of <i>P. splendens</i>.</p

The gene expression of D12 and D9 based on the data of DGE among different fermentation stages of <i>P. splendens</i>.

<p>The gene expression of D12 and D9 based on the data of DGE among different fermentation stages of <i>P. splendens</i>.</p

Scaffolds length distribution of <i>P. splendens</i>.

<p>Scaffolds length distribution of <i>P. splendens</i>.</p

Effect of query sequence length on the percentage of sequences for which significant matches are found.

<p>The proportion of sequences with matches in NCBI nr databases is greater among the longer assembled sequences (with a cut-off E-value of 10⁻⁵).</p

Numbers of DGE unigenes in each comparison (2-1: 48 h, 4-1: 100 h, 6-1: 148 h).

<p>Up-(red) and down-regulated (green) unigenes were quantified.</p