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.     

The Salinity Tolerance of Freshwater Cyanobacterium Synechococcus sp. PCC 7942 is Determined by Its Ability for Osmotic Adjustment and Presence of Osmolyte Sucrose

We investigated the factors that impose an upper limit of salinity tolerance to the unicellular freshwater cyanobacterium Synechococcus sp. PCC 7942. Above approx. 0.4 M NaCl, Synechococcus cells cease to proliferate, after having accumulated 0.3 M sucrose. Cells that pre-accumulated sucrose could tolerate up to 0.5 M NaCl, but not 0.6 M NaCl. After exposure to 0.5 M NaCl or higher, the cells were irreversibly modified becoming unable for osmotic volume adjustments. This revised version was published online in August 2006 with corrections to the Cover Date.     

A method to probe the cytoplasmic osmolality and osmotic water and solute fluxes across the cell membrane of cyanobacteria with chlorophyll a fluorescence: Experiments with Synechococcus sp. PCC7942

We present a method with which osmotic properties of the cytoplasm of cyanobacterial cells and the osmotic permeability of plasma membranes to water and solutes can be assessed from measurements of chlorophyll a fluorescence. When the electron transport of photosystem II is inhibited, the quantum yield of chlorophyll a fluorescence in cyanobacterial cells varied between a low yield limit that was attained after acclimation to darkness (state 2) and a high yield limit that was attained after acclimation to light (state 1). It was shown recently that the difference between chlorophyll a fluorescence of light‐acclimated and of dark‐acclimated cells relates quantitatively to the internal osmolality of cyanobacteria (G. C. Papageorgiou and A. Alygizaki‐Zorba. 1997. Biochim. Biophys. Acta 1335: 1‐4). In the present work we employed rapid mixing of Synechococcus sp. PCC7942 (strain PAMCOD) suspensions with solutions of defined osmolality in order to measure cell osmolality and turgor threshold, as well as water and solute fluxes across cell membranes. Concentration upshocks with sorbitol, glycine betaine, Na⁺ and K⁺ salts caused rapid (t_1/2 < 10 ms) depression of fluorescence that was correlated to osmotic water outflow from the cells. The fluorescence remained depressed in all cases except for NaCl. With NaCl, the depression was transient and fluorescence recovered with an apparent time constant of 200 ms. The fluorescence rise correlates to inflows of NaCl and water.     

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.     

LiDAR‐derived canopy structure supports the more‐individuals hypothesis for arthropod diversity in temperate forests

Despite considerable progress in the ability to measure the complex 3‐D structure of forests with the improvement of remote‐sensing techniques, our mechanistic understanding of how biodiversity is linked to canopy structure is still limited. Here we tested whether the increase in arthropod abundance and richness in beech forest canopies with increasing canopy complexity supports the more‐individuals hypothesis or the habitat‐heterogeneity hypothesis. We used fogging to collect arthropod samples from 80 standardized plots from canopies of single‐ to multi‐layered mature montane European beech stands. Tree height and an independent measure of vertical heterogeneity – the vertical distribution ratio – on each arthropod sampling plot were derived from high‐resolution full‐waveform airborne laser scanning data. Mixed‐model path analysis based on almost 20 000 specimens of 762 species from 11 orders provided support for the more‐individuals hypothesis, with higher arthropod abundance but not higher species richness in stands with a more equal vertical distribution of plant biomass. By contrast, we found no support for the habitat‐heterogeneity hypothesis. The increase in the number of individuals with increasing vertical distribution of biomass might be caused either by increasing leaf area, as indicated by higher space filling and productivity in multi‐layered stands, or by higher persistence of arthropod populations owing to better shelter, reduced competition and more refuges under harsh conditions, or by both. High‐resolution airborne laser scanning, with its ability to penetrate dense canopies under leaf‐on conditions, has proved suitable for measuring vertical structures as a predictor for canopy diversity. Expanding combinations of remote‐sensing and canopy‐biodiversity data opens many avenues for improving our understanding of the link between diversity and forest structures.     

Linearized oscillations in population dynamics

A linearized oscillation theorem due to Kulenović, Ladas and Meimaridou (1987,Quart. appl. Math. XLV, 155–164) and an extension of it are applied to obtain the oscillation of solutions of several equations which have appeared in population dynamics. They include the logistic equation with several delays, Nicholson's blowflies model as described by Gurney, Blythe and Nisbet (1980,Nature, Lond. 287, 17–21) and the Lasota-Wazewska model of the red blood cell supply in an animal. We also developed a linearized oscillation result for difference equations and applied it to several equations taken from the biological literature.     

Structure of tandem RNA recognition motifs from polypyrimidine tract binding protein reveals novel features of the RRM fold

Polypyrimidine tract binding protein (PTB), an RNA binding protein containing four RNA recognition motifs (RRMs), is involved in both pre-mRNA splicing and translation initiation directed by picornaviral internal ribosome entry sites. Sequence comparisons previously indicated that PTB is a non-canonical RRM protein. The solution structure of a PTB fragment containing RRMs 3 and 4 shows that the protein consists of two domains connected by a long, flexible linker. The two domains tumble independently in solution, having no fixed relative orientation. In addition to the betaalphabetabetaalphabeta topology, which is characteristic of RRM domains, the C-terminal extension of PTB RRM-3 incorporates an unanticipated fifth beta-strand, which extends the RNA binding surface. The long, disordered polypeptide connecting beta4 and beta5 in RRM-3 is poised above the RNA binding surface and is likely to contribute to RNA recognition. Mutational analyses show that both RRM-3 and RRM-4 contribute to RNA binding specificity and that, despite its unusual sequence, PTB binds RNA in a manner akin to that of other RRM proteins.     

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).