Involvement of the C-terminal part of Pseudomonas fluorescens OprF in the modulation of its pore-forming properties

The major outer-membrane protein, OprF, from the psychrotrophic bacterium Pseudomonas fluorescens undergoes a reduction of its conductance value (from 250 pS to 80 pS) when the growth temperature is shifted from 28 degrees C to 8 degrees C. The involvement of changes in tertiary or quaternary structure in this behaviour, was implied by enzymatic digestion experiments in which OprFs purified from 8 degrees C and 28 degrees C cultures showed different accessibility to pronase. Resistant proteolytic fragments of 19 kDa, obtained from both OprF preparations, were identified as the N-terminal half of the native protein. These 19 kDa fragments induced ion channels in planar lipid bilayers with similar conductance values of 65-75 pS in 1 M NaCl, in contrast to the native proteins. Thus, the C-terminal part of the protein is required for the growth temperature-dependent modulation of OprF channel-forming properties. LPS was not detected on the proteolytic fragments while it was found in similar amounts on the native OprFs. These results suggest the LPS/porin association occurs through the C-terminal part of the porin. Radiolabelling experiments showed different phosphorylation levels of LPS for 8 degrees C and 28 degrees C cultures. Thus, in response to growth temperature, the structural modification of the LPS could be associated to the modulation of OprF pore size.     

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.     

Evolutionary rescue by compensatory mutations is constrained by genomic and environmental backgrounds

Since deleterious mutations may be rescued by secondary mutations during evolution, compensatory evolution could identify genetic solutions leading to therapeutic targets. Here, we tested this hypothesis and examined whether these solutions would be universal or would need to be adapted to one's genetic and environmental makeups. We performed experimental evolutionary rescue in a yeast disease model for the Wiskott–Aldrich syndrome in two genetic backgrounds and carbon sources. We found that multiple aspects of the evolutionary rescue outcome depend on the genotype, the environment, or a combination thereof. Specifically, the compensatory mutation rate and type, the molecular rescue mechanism, the genetic target, and the associated fitness cost varied across contexts. The course of compensatory evolution is therefore highly contingent on the initial conditions in which the deleterious mutation occurs. In addition, these results reveal biologically favored therapeutic targets for the Wiskott–Aldrich syndrome, including the target of an unrelated clinically approved drug. Our results experimentally illustrate the importance of epistasis and environmental evolutionary constraints that shape the adaptive landscape and evolutionary rate of molecular networks.     

Novel Use of PIT Tags in Sea Cucumbers: Promising Results with the Commercial Species Cucumaria frondosa

The lack of a reliable and innocuous mark-recapture method has limited studies that would provide essential information for the management of commercial sea cucumbers. Tagging sea cucumbers is notoriously difficult because of their plastic nature and autolysis capacities. The markers that have so far been tested, mainly on or through the body wall, were either lost rapidly or had major drawbacks (e.g. suitable only for batch identification, requiring complex analysis, causing infections, necrosis, behavioural changes and mortality). The present study explored the efficacy of passive integrated transponder (PIT) tags for individually marking sea cucumbers by assessing retention rates and long-term side effects of tags inserted in previously unstudied tissues/organs. Individuals of the species Cucumaria frondosa were tagged in the body wall, aquapharyngeal bulb and at the base of the oral tentacles. They were monitored closely for evidence of stress, infection, change in feeding and spawning behaviour and tag retention rate. Implanting the tag in an oral tentacle to reach the hydrovascular system of the aquapharyngeal bulb achieved the best retention rates in full-size individuals: from a maximum of 92% after 30 days to 68% at the end of the experimental period (300 days). Efficacy was lower in smaller individuals (84% after 30 d and 42% after 300 d). Following a slight increase in cloacal movements for 15 h post tagging, no side effect was noted in sea cucumbers tagged in the aquapharyngeal bulb via the tentacles. Feeding and spawning behaviours were not affected and no signs of infections or abnormal cell development in the vicinity of the tags were observed. This study indicates that marking sea cucumbers with 8.2 mm long PIT tags implanted via the oral tentacle is an effective technique, yielding relatively high retention rates over long periods without any detectable physiological or behavioural effects.     

Complex offspring size effects: variations across life stages and between species

Classical optimality models of offspring size and number assume a monotonically increasing relationship between offspring size and performance. In aquatic organisms with complex life cycles, the size–performance function is particularly hard to grasp because measures of performance are varied and their relationships with size may not be consistent throughout early ontogeny. Here, we examine size effects in premetamorphic (larval) and postmetamorphic (juvenile) stages of brooding marine animals and show that they vary contextually in strength and direction during ontogeny and among species. Larger offspring of the sea anemone Urticina felina generally outperformed small siblings at the larval stage (i.e., greater settlement and survival rates under suboptimal conditions). However, results differed when analyses were conducted at the intrabrood versus across‐brood levels, suggesting that the relationship between larval size and performance is mediated by parentage. At the juvenile stage (15 months), small offspring were less susceptible than large ones to predation by subadult nudibranchs and both sizes performed similarly when facing adult nudibranchs. In a sympatric species with a different life history (Aulactinia stella), all juveniles suffered similar predation rates by subadult nudibranchs, but smaller juveniles performed better (lower mortalities) when facing adult nudibranchs. Size differences in premetamorphic performance of U. felina were linked to total lipid contents of larvae, whereas size‐specific predation of juvenile stages followed the general predictions of the optimal foraging strategy. These findings emphasize the challenge in gathering empirical support for a positive monotonic size–performance function in taxa that exhibit complex life cycles, which are dominant in the sea.     

Symmetrical α-bromoacryloylamido diaryldienone derivatives as a novel series of antiproliferative agents. Design,synthesis and biological evaluation

In a continuing study of hybrid compounds containing the α-bromoacryloyl moiety as potential anticancer drugs, we synthesized a novel series of hybrids 4a–h, in which this moiety was linked to a 1,5-diaryl-1,4-pentadien-3-one system. Many of the conjugates prepared (4b, 4c, 4e and 4g) demonstrated pronounced, submicromolar antiproliferative activity against four cancer cell lines. Moreover, compound 4b induced apoptosis through the mitochondrial pathway and activated caspase-3 in a concentration-dependent manner.     

Diversity and Functionality of Arbuscular Mycorrhizal Fungi in Three Plant Communities in Semiarid Grasslands National Park, Canada

Septate endophytes proliferating in the roots of grasslands’ plants shed doubts on the importance of arbuscular mycorrhizal (AM) symbioses in dry soils. The functionality and diversity of the AM symbioses formed in four replicates of three adjacent plant communities (agricultural, native, and restored) in Grasslands National Park, Canada were assessed in periods of moisture sufficiency and deficiency typical of early and late summer in the region. The community structure of AM fungi, as determined by polymerase chain reaction-denaturing gradient gel electrophoresis, varied with sampling time and plant community. Soil properties other than soil moisture did not change significantly with sampling time. The DNA sequences dominating AM extraradical networks in dry soil apparently belonged to rare taxa unreported in GenBank. DNA sequences of Glomus viscosum, Glomus mosseae, and Glomus hoi were dominant under conditions of moisture sufficiency. In total, nine different AM fungal sequences were found suggesting a role for the AM symbioses in semiarid areas. Significant positive linear relationships between plant P and N concentrations and active extraradical AM fungal biomass, estimated by the abundance of the phospholipid fatty acid marker 16:1ω5, existed under conditions of moisture sufficiency, but not under dry conditions. Active extraradical AM fungal biomass had significantly positive linear relationship with the abundance of two early season grasses, Agropyron cristatum (L.) Gaertn. and Koeleria gracilis Pers., but no relationship was found under dry conditions. The AM symbioses formed under conditions of moisture sufficiency typical of early summer at this location appear to be important for the nutrition of grassland plant communities, but no evidence of mutualism was found under the dry conditions of late summer.     

Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation

Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus.     

Chitooligosaccharide sensing and downstream signaling: contrasted outcomes in pathogenic and beneficial plant–microbe interactions

In plants, short chitin oligosaccharides and chitosan fragments (collectively referred to as chitooligosaccharides) are well-known elicitors that trigger defense gene expression, synthesis of antimicrobial compounds, and cell wall strengthening. Recent findings have shed new light on chitin-sensing mechanisms and downstream activation of intracellular signaling networks that mediate plant defense responses. Interestingly, chitin receptors possess several lysin motif domains that are also found in several legume Nod factor receptors. Nod factors are chitin-related molecules produced by nitrogen-fixing rhizobia to induce root nodulation. The fact that chitin and Nod factor receptors share structural similarity suggests an evolutionary conserved relationship between mechanisms enabling recognition of both deleterious and beneficial microorganisms. Here, we will present an update on molecular events involved in chitooligosaccharide sensing and downstream signaling pathways in plants and will discuss how structurally related signals may lead to such contrasted outcomes during plant–microbe interactions.