Reappraisal of early Miocene rails (Aves,Rallidae) from central France: diversity and character evolution

In Europe, Miocene rails (Aves, Rallidae) are quite abundant, but their phylogenetic placement in the context of recent forms has remained elusive. Rails from the early Miocene of the Saint‐Gérand‐le‐Puy area in central France were first described in the 19th century, and currently, only two species are recognized, namely Palaeoaramides christyi and Paraortygometra porzanoides. Our examination of the material however suggests the presence of four, likely coeval, species of rail from these deposits. Palaeoaramides eximius, previously synonymized with Palaeoaramides christyi, is here shown to probably be a distinct species, and a previously unrecognized rail, Baselrallus intermedius gen. et sp. nov., is described. To find out how these fossil rails are related to modern Rallidae, we compared them with an extensive sample of extant rails and identified plesiomorphic and derived features for crown group Rallidae. Our assessment does not support a particularly close relationship of either Palaeoaramides to Aramides or Paraortygometra to Crex (Ortygometra), and overall, these fossil rails are more primitive than previously assumed. Based on our observations of the morphology of the previously undescribed humerus of Palaeoaramides, we show this taxon to be outside crown group Rallidae, and perhaps closely related to the early Oligocene taxon Belgirallus. On the other hand, Paraortygometra porzanoides bears a resemblance to recent flufftails (Sarothrura spp.) in some elements, but whether it can be included in a clade together with flufftails is uncertain.     

Volatile exposure within the honeybee hive and its effect on olfactory discrimination

Honeybees of different ages and reproductive castes cohabit in the hive where they are exposed to many odors that might affect associative learning. Our aim was to analyze the role of odors pre-exposed as volatiles on appetitive learning in honeybees of different ages and search for their long-term effect both under natural and laboratory conditions. By evaluating memory acquisition and retention through a differential proboscis extension response conditioning, we found that hive-exposed odors offered as a reinforced conditioned stimulus during training promoted a learning-reduced effect [latent inhibition (LI)]. On the other hand, no effect was found when the non-reinforced conditioned stimulus was pre-exposed. The LI effect varied with the odor identity. However, only slight differences were found with the age of the bees. Exposure-conditioning intervals longer than 24 h did not show an LI effect unless the odor concentration was increased or exposure was prolonged. Our results show that pre-exposed volatiles could either reduce learning performance, if this odor is later associated with food, or be irrelevant in the case that alternative scented resources circulate within the colony. The differential effects found according to the olfactory exposure characteristics could strongly influence the propagation of chemosensory information within the hive.     

Preparation of Chloramphenicol/Amino Acid Combinations Exhibiting Enhanced Dissolution Rates and Reduced Drug-Induced Oxidative Stress

Chloramphenicol is an old antibiotic agent that is re-emerging as a valuable alternative for the treatment of multidrug-resistant pathogens. However, it exhibits suboptimal biopharmaceutical properties and toxicity profiles. In this work, chloramphenicol was combined with essential amino acids (arginine, cysteine, glycine, and leucine) with the aim of improving its dissolution rate and reduce its toxicity towards leukocytes. The chloramphenicol/amino acid solid samples were prepared by freeze-drying method and characterized in the solid state by using Fourier transform infrared spectroscopy, powder X-ray diffraction, differential scanning calorimetry, scanning electron microscopy, and solid-state nuclear magnetic resonance. The dissolution properties, antimicrobial activity, reactive oxygen species production, and stability of the different samples were studied. The dissolution rate of all combinations was significantly increased in comparison to that of the pure active pharmaceutical ingredient. Additionally, oxidative stress production in human leukocytes caused by chloramphenicol was decreased in the chloramphenicol/amino acid combinations, while the antimicrobial activity of the antibiotic was maintained. The CAP:Leu binary combination resulted in the most outstanding solid system makes it suitable candidate for the development of pharmaceutical formulations of this antimicrobial agent with an improved safety profile.     

Structure analysis of the Staphylococcus aureus UDP-N-acetyl-mannosamine dehydrogenase Cap5O involved in capsular polysaccharide biosynthesis

Bacterial UDP-sugar dehydrogenases are part of the biosynthesis pathway of extracellular polysaccharides. These compounds act as important virulence factors by protecting the cell from opsonophagocytosis and complement-mediated killing. In Staphylococcus aureus, the protein Cap5O catalyzes the oxidation of UDP-N-acetyl-mannosamine to UDP-N-acetyl-mannosaminuronic acid. Cap5O is crucial for the production of serotype 5 capsular polysaccharide that prevents the interaction of bacteria with both phagocytic and nonphagocytic eukaryotic cells. However, details of its catalytic mechanism remain unknown. We thus crystallized Cap5O and solved the first structure of an UDP-N-acetyl-mannosamine dehydrogenase. This study revealed that the catalytic cysteine makes a disulfide bond that has never been observed in other structurally characterized members of the NDP-sugar dehydrogenase family. Biochemical and mutagenesis experiments demonstrated that the formation of this disulfide bridge regulates the activity of Cap5O. We also identified two arginine residues essential for Cap5O activity. Previous data suggested that Cap5O is activated by tyrosine phosphorylation, so we characterized the phosphorylation site and examined the underlying regulatory mechanism.     

Season effect on genitalia and epididymal sperm from Iberian red deer, roe deer and Cantabrian chamois

Seasonality deeply affects the physiology and behavior of many species, and must be taken into account when biological resource banks (BRBs) are established. We have studied the effect of seasonality on many reproductive parameters of free-ranging Iberian red deer, roe deer and Cantabrian chamois, living in Spain. Testicles from hunted animals were collected and sent to our laboratory at different times during the year. We recorded the weight and volume of testis, the weight of the epididymis and its separate parts (caput, corpus, and cauda), the weight of the sperm sample collected from the cauda epididymis, and several sperm parameters (sperm concentration, spermatozoa recovered, motility, HOS test reactivity, acrosomal status, and viability). We studied the data according to several periods, defined accordingly to each species. For red deer, we defined rut (mid-September to mid-October), post-rut (mid-October to mid-December), and non-breeding season (February). For roe deer, they were pre-rut (June), rut (July), post-rut (first fortnight of August), and non-breeding season (September). For chamois: non-breeding season (June to mid-September) and breeding season (October-November). The rut/breeding season yielded significantly higher numbers for almost all parameters. However, in the case of red deer, sperm quality was higher in the post-rut. For roe deer, testicular weight was similar in the pre-rut and in the rut, and sperm quality did not differ significantly between these two periods, although we noticed higher values in the rut. In the case of chamois, sperm quality did not differ significantly from the breeding season, but data distribution suggested that in the non-breeding season there are less males with sperm of good quality. On the whole, we find these results of interest for BRB planning. The best season to collect sperm in this species would be the breeding season. However, post-rut in red deer, pre-rut in roe deer, and non-breeding season in chamois could be used too, because of the acceptable sperm quality, despite the lower quantity salvaged. More in-depth research needs to be carried out on the quality of sperm salvaged at different times of the year in order to confirm these findings.     

Temperate Snake Community in South America: Is Diet Determined by Phylogeny or Ecology?

Communities are complex and dynamic systems that change with time. The first attempts to explain how they were structured involve contemporary phenomena like ecological interactions between species (e.g., competition and predation) and led to the competition-predation hypothesis. Recently, the deep history hypothesis has emerged, which suggests that profound differences in the evolutionary history of organisms resulted in a number of ecological features that remain largely on species that are part of existing communities. Nevertheless, both phylogenetic structure and ecological interactions can act together to determine the structure of a community. Because diet is one of the main niche axes, in this study we evaluated, for the first time, the impact of ecological and phylogenetic factors on the diet of Neotropical snakes from the subtropical-temperate region of South America. Additionally, we studied their relationship with morphological and environmental aspects to understand the natural history and ecology of this community. A canonical phylogenetical ordination analysis showed that phylogeny explained most of the variation in diet, whereas ecological characters explained very little of this variation. Furthermore, some snakes that shared the habitat showed some degree of diet convergence, in accordance with the competition-predation hypothesis, although phylogeny remained the major determinant in structuring this community. The clade with the greatest variability was the subfamily Dipsadinae, whose members had a very different type of diet, based on soft-bodied invertebrates. Our results are consistent with the deep history hypothesis, and we suggest that the community under study has a deep phylogenetic effect that explains most of the variation in the diet.     

Molecular characterization of inulosucrase from Leuconostoc citreum: a fructosyltransferase within a glucosyltransferase

The gene coding for inulosucrase in Leuconostoc citreum CW28, islA, was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme catalyzed inulin synthesis from sucrose like the wild-type enzyme. Inulosucrase presents an unusual structure: its N-terminal region is similar to the variable region of glucosyltransferases, its catalytic domain is similar to fructosyltransferases from various microorganisms, and its C-terminal domain presents similarity to the glucan binding domain from alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355. From sequence comparison, it was found that this fructosyltransferase is a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase by a fructosyltransferase. Two different forms of the islA gene truncated in the C-terminal glucan binding domain were successfully expressed in E. coli and retained their ability to synthesize inulin but lost thermal stability. This is the first report of an inulosucrase bearing structural features of both glucosyltransferases and fructosyltransferases.     

Relevance of Fatty Acid Covalently Bound to Escherichia coli ��-Hemolysin and Membrane Microdomains in the Oligomerization Process

α-Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of Escherichia coli that causes lysis of several mammalian cells, including erythrocytes of different species. HlyA is synthesized as a protoxin, pro-HlyA, which is activated by acylation at two internal lysines Lys-563 and Lys-689. It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes, but these experiments have yielded conflicting results about the structure of the pore. In this study, HlyA cysteine replacement mutant proteins of amino acids have been labeled with Alexa-488 and Alexa-546. Fluorescence resonance energy transfer measurements, employing labeled toxin bound to sheep ghost erythrocytes, have demonstrated that HlyA oligomerizes on erythrocyte membranes. As the cytotoxic activity is absolutely dependent on acylation, we have studied the role of acylation in the oligomerization, demonstrating that fatty acids are essential in this process. On the other hand, fluorescence resonance energy transfer and the hemolytic activity decrease when the erythrocyte ghosts are cholesterol-depleted, hence indicating the role of membrane microdomains in the clustering of HlyA. Simultaneously, HlyA was found in detergent-resistant membranes. Pro-HlyA has also been found in detergent-resistant membranes, thus demonstrating that the importance of acyl chains in toxin oligomerization is the promotion of protein-protein interaction. These results change the concept of the main role assigned to acyl chain in the targeting of proteins to membrane microdomains.Escherichia coli α-hemolysin, HlyA,⁴ is an exotoxin that elicits a number of responses from mammalian target cells and also alters the membrane permeability of host cells, causing lysis and death (1, 2). Synthesis, maturation, and secretion of E. coli HlyA are determined by the hlyCABD operon (3). The gene A product is a 110-kDa polypeptide corresponding to protoxin (Pro-HlyA), which is matured in bacterial cytosol to the active form (HlyA) by HlyC-directed acylation. This post-translational modification involves a covalent amide linkage of fatty acids at two internal lysine residues (Lys-563 and Lys-689) for activation (4). HlyA activated in vivo consists of a heterogeneous family of up to nine different covalent structures (two acylation sites and three possible modifying groups in each site, C14:0 (68%), C15:0 (26%) and C17:0 (6%) (5)). Although these fatty acids are not required for the binding of the toxin to membranes, they are essential for the hemolytic process, inducing a molten globule conformation and promoting the irreversibility of the binding (6, 7).It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes. However, these experiments have yielded conflicting results. Although a group of researchers is in favor of a monomer as the active species of the toxin in membranes, other groups postulate that an oligomerization process is involved. Based on experiments with lipid bilayers, Menestrina et al. (8) have suggested that one single HlyA molecule is responsible for the formation of the channel. HlyA has also been recovered from deoxycholate-solubilized erythrocyte membranes as a monomer, indicating either that oligomerization is not required for pore formation or that oligomers are dissociated in the detergent (1).On the other hand, Benz et al. (9) have found that small variations of toxin concentration have had a considerable effect on the specific membrane conductance. An increase in HlyA concentration, by a factor of 5, results in about 40–100-fold higher membrane conductance. This means that several HlyA molecules could be involved in channel formation (9). Besides, they have found that the active channel-forming oligomer and inactive monomer are in an association-dissociation equilibrium (10). In addition, the complementation of inactive deleted mutant proteins of HlyA with the corresponding wild type toxin produces hemolytic activity, suggesting that two or more toxin molecules aggregate before pore formation (11). All of the evidence suggests the formation of an oligomer.Experiments employing erythrocytes and model membranes have shown that the lesion created by HlyA is perhaps a more complicated event than the creation of a simple, static protein-lined pore. We have recently found that addition of nanomolar concentrations of toxin to planar lipid membranes have resulted in a decrease in membrane lifetime up to 3 orders of magnitude in a voltage-dependent manner, a typical behavior of proteolipidic pores (12). Moayeri and Welch (13) have previously demonstrated that osmotic protection of erythrocytes by sugars of different sizes is a function of toxin concentration and assay time. It appears that HlyA induces heterogeneous erythrocyte lesions that increase in size over time and that the rate of the putative growth in the size of HlyA-mediated lesions is temperature-dependent (13).On the other hand, it has been recognized that a variety of pathogens and toxins interacts with microdomains in the plasma membrane. These microdomains are enriched in cholesterol and sphingolipids and probably exist in a liquid-ordered phase, in which lipid acyl chains are extended and ordered (14). Many proteins are targeted to these membrane microdomains by their favorable association with ordered lipids. Interestingly, these proteins are linked to saturated acyl chains, which partition well into these domains (15).In this context, and in view of the fact that acyl chains covalently bound to proteins are determinant of specific protein-protein interactions, this research presents a study of HlyA oligomerization on sheep erythrocytes, as well as the implication of fatty acids and cholesterol-enriched microdomains in this process.