Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Identification of a discrete intermediate in the assembly/disassembly of physalis mottle tymovirus through mutational analysis.

Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and deletion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the assembly of the virus and to identify intermediates in this process. N-terminal 30, 34, 35 and 39 amino acid deletion and single C-terminal (N188) deletion mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also constructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T=3 particles in vivo while deletions of 34, 35 and 39 amino acid residues resulted in the mutant proteins that were insoluble. Interestingly, the coat protein (pR PhCP) expressed using pRSET B vector with an additional 41 amino acid residues at the N terminus also assembled into T=3 particles that were more compact and had a smaller diameter. These results demonstrate that the amino-terminal segment is flexible and either the deletion or addition of amino acid residues at the N terminus does not affect T=3 capsid assembly. In contrast, the deletion of even a single residue from the C terminus (PhN188Delta1) resulted in capsids that were unstable. These capsids disassembled to a discrete intermediate with a sedimentation coefficent of 19.4 S. However, the replacement of C-terminal asparagine 188 by alanine led to the formation of stable capsids. The C75A and D144N mutant proteins also assembled into capsids that were as stable as the pR PhCP, suggesting that C75 and D144 are not crucial for the T=3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant proteins failed to form capsids and were present as heterogeneous aggregates. Interestingly, the pR PhK143E mutant protein behaved in a manner similar to the C-terminal deletion protein in forming unstable capsids. The intermediate with an s value of 19.4 S was the major assembly product of pR PhH69A mutant protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188Delta1, pR PhH69A and pR PhK143E mutant proteins.     

Enantioselective metabolism of primaquine by human CYP2D6

Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated. Six diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (Hss LDH) or plasmodial lactate dehydrogenase (Pf LDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro. All tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of Pf LDH; specificity between the residues involved in H-bonds of the Pf LDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of Pf LDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive. The presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than Pf LDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.     

Synthesis and pharmacological evaluation of aryl aminosulfonamide derivatives as potent 5-HT6 receptor antagonists

A series of novel aryl aminosulfonamides was designed and synthesized as 5-HT₆ receptor ligands. Many compounds screened in a functional reporter gene based assay displayed potent antagonistic activity with Kb values in the range of 0.02–10 nM. The lead compound 11m exemplified in this series showed good ADME surrogate properties, acceptable pharmacokinetic profile and is active in animal models of cognition like novel object recognition test and Morris water maze. The compound was selected for detailed profiling.     

The effect of acidification and the combined effects of acidification/lipid extraction on carbon stable isotope ratios for sub-arctic and arctic marine zooplankton species

Stable isotope ratios of carbon (δ ¹³C) in zooplankton are widely applied in ecosystem-level studies of energy flux and trophic interactions. Carbonate (CaCO₃) and lipid content are highly variable both among and within zooplankton species. Such variability can arise from the δ ¹³C-depleted nature of lipids as well as differences in carbon incorporation among tissues (e.g., relative amount of carbonate). Critically, the impact of the common lipid- and carbonate-normalization steps of extraction and acidification is poorly understood and applied in an inconsistent manner. Here, we investigated the effect of lipid extraction and sample acidification (CaCO₃ removal) on δ ¹³C in sub-arctic and arctic marine zooplankton species. Our results indicate that, with the exception of the shelled mollusc Limacina helicina, acidification of samples can be omitted for all other marine zooplankton considered in this study. In the case of L. helicina, δ ¹³C can be corrected for carbonate content using the linear equation developed in this study. In contrast, the δ ¹³C for all species was significantly enriched by a combination of lipid extraction and acidification (up to +4.9 ‰) prior to stable isotope analysis. Our data were used to develop simple, predictive species-specific correction models for lipid-acid-normalized δ ¹³C using C:N and/or untreated δ ¹³C values. Our results indicate that the δ ¹³C value for all species, including those with lower C:N ratios (~3–4), should be corrected for lipid content. We recommend lipid extraction whenever possible, or else the use of species/taxon-specific δ ¹³C lipid-normalization models for accurate determination of carbon sources and dynamics for arctic and sub-arctic marine zooplankton.     

Prey size diversity hinders biomass trophic transfer and predator size diversity promotes it in planktonic communities

Body size exerts multiple effects on plankton food-web interactions. However, the influence of size structure on trophic transfer remains poorly quantified in the field. Here, we examine how the size diversity of prey (nano-microplankton) and predators (mesozooplankton) influence trophic transfer efficiency (using biomass ratio as a proxy) in natural marine ecosystems. Our results support previous studies on single trophic levels: transfer efficiency decreases with increasing prey size diversity and is enhanced with greater predator size diversity. We further show that communities with low nano-microplankton size diversity and high mesozooplankton size diversity tend to occur in warmer environments with low nutrient concentrations, thus promoting trophic transfer to higher trophic levels in those conditions. Moreover, we reveal an interactive effect of predator and prey size diversities: the positive effect of predator size diversity becomes influential when prey size diversity is high. Mechanistically, the negative effect of prey size diversity on trophic transfer may be explained by unicellular size-based metabolic constraints as well as trade-offs between growth and predation avoidance with size, whereas increasing predator size diversity may enhance diet niche partitioning and thus promote trophic transfer. These findings provide insights into size-based theories of ecosystem functioning, with implications for ecosystem predictive models.     

Protein biomarkers for in vitro testing of embryotoxicity

There are new challenges for hazard and risk assessment in the chemical industry with regard to REACH legislation in Europe and related activities in the U.S. and Japan, which require the development of novel in vitro models for the molecular characterization of drug- or chemical-related effects replacing conventional animal testing. In the frame of a European FP6 project on reproductive toxicology ( www.reprotect.eu ), we prepared protein samples from mouse embryonic stem cells differentiated into contracting cardiomyocytes according to the validated embryonic stem cell test (EST) protocol, which had been exposed to toxic substances selected by an expert committee from different in vivo categories of embryotoxicity. Lysates were used to carry out the following investigations: (i) identify optimal dose range conditions in the EST that are suitable for (ii) performing a differential quantitative proteomic study of underlying molecular pathways, (iii) define classes of substances with similar proteomic response patterns, (iv) relate these classes to the traditional in vivo categories of embryotoxicity with (v) the final goal to identify novel surrogate protein biomarker candidates for embryo toxicity. We found two distinct classes of toxic substances (Dinoseb, Ochratoxin-A, and Nitrofen vs β-aminoproprionitril, Metoclopramide, Doxylamine succinate, and d-penicillamine) with clear pathway-related differences in their proteomic patterns. Most notably, different responses to cluster 1 and cluster 2 substances were observed for Heat shock protein β-1, Ras-GTPase-activating protein SH3-domain binding protein, Ran binding protein 5, and Calreticulin, Dihydropyrimidinase-like 2 (Ulip2 protein). On the other hand, Heat shock protein 8 and Fscn1 protein were down-regulated by all compounds from both clusters.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).