Using evolutionary information and ancestral sequences to understand the sequence-function relationship in GLP-1 agonists

Glucagon-like peptide-1 (GLP-1) is an incretin hormone with therapeutic potential for type 2 diabetes. A variety of GLP-1 sequences are known from amphibian species, and some of these have been tested here and found to be able to bind and activate the human GLP-1 receptor. While little difference was observed for the in vitro potency for the human GLP-1 receptor, larger differences were found in the enzymatic stability of these peptides. Two peptides showed increased enzymatic stability, and they group together phylogenetically, though they originate from Amphibia and Reptilia. We have used ancestral sequence reconstruction to analyze the evolution of these GLP-1 molecules, including the synthesis of new peptides. We find that the increased stability could not be observed in the resurrected peptides from the common ancestor of frogs, even though they maintain the ability to activate the human GLP-1 receptor. Another method, using residue mapping on evolutionary branches yielded peptides that had maintained potency towards the receptor and also showed increased stability. This represents a new approach using evolutionary data in protein engineering.     

A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms

Pseudomonas aeruginosa produces extracellular DNA which functions as a cell-to-cell interconnecting matrix component in biofilms. Comparison of extracellular DNA and chromosomal DNA by the use of polymerase chain reaction and Southern analysis suggested that the extracellular DNA is similar to whole-genome DNA. Evidence that the extracellular DNA in P. aeruginosa biofilms and cultures is generated via lysis of a subpopulation of the bacteria was obtained through experiments where extracellular beta-galactosidase released from lacZ-containing P. aeruginosa strains was assessed. Experiments with the wild type and lasIrhlI, pqsA, pqsL and fliMpilA mutants indicated that the extracellular DNA is generated via a mechanism which is dependent on acyl homoserine lactone and Pseudomonas quinolone signalling, as well as on flagella and type IV pili. Microscopic investigation of flow chamber-grown wild-type P. aeruginosa biofilms stained with different DNA stains suggested that the extracellular DNA is located primarily in the stalks of mushroom-shaped multicellular structures, with a high concentration especially in the outer part of the stalks forming a border between the stalk-forming bacteria and the cap-forming bacteria. Biofilms formed by lasIrhlI, pqsA and fliMpilA mutants contained less extracellular DNA than biofilms formed by the wild type, and the mutant biofilms were more susceptible to treatment with sodium dodecyl sulphate than the wild-type biofilm.     

PCR-RFLP of ITS rDNA for the rapid identification of Penicillium subgenus Biverticillium species

RFLP of ITS rDNA is proposed as a useful tool for molecular identification of the most common species of biverticillate penicillia. 60 isolates were analysed representing 13 species and 21 unique sequences were produced. The combination of five restriction enzymes was successful in separating 12 species. However, the variety Penicillium purpurogenum var. rubrisclerotium remained indistinguishable from Penicillium funiculosum. P. funiculosum appeared as the most confused species, being mis-identified with Penicillium miniolutum and Penicillium pinophilum, which were originally part of the species, and with P. purpurogenum perhaps because of the common production of red pigment. Penicillium variabile was difficult to investigate as introns were found on half of the isolates. Penicillium piceum, Penicillium rugulosum, Penicillium loliense, Penicillium erythromellis and P. purpurogenum were homogeneous from molecular and morphological positions and corresponded to a well circumscribed taxon. Furthermore, intraspecific variability was evidenced within P. pinophilum and P. funiculosum. The ex-type isolate of P. funiculosum produced a unique pattern. The method is sensitive, rapid and inexpensive and can be used for isolate identification of the biverticillate species. It is recommended particularly when many isolates have to be authentificated prior to analysis for phylogenetic assessment or population genetics.     

Co-occurring graminoid and forb species do not differ in their root morphological response to soil heterogeneity

We measured the proliferation of roots into experimental nutrient patches in a grassland community, distinguishing roots of graminoids and forbs. Biomass, length, and specific length were estimated for roots of each of the two functional groups, collected from patches differing in nutrient concentration, and established at four different times during a season. The ratio of graminoid and forb roots was compared with the graminoid-forb ratio in the above-ground biomass. Plant roots proliferated more intensively into patches with higher nutrient concentration, but the roots of the two functional groups had a similar ability to target richer patches. Relative proportion of graminoids was higher below-ground than above-ground and changed during the season, being lowest after mowing. Specific root length was higher for graminoid species, but did not respond to nutrient concentration in patches for either functional group. This is the first study to provide comparative information about root morphological response for graminoids and forbs, measured in a real, semi-natural plant community. The study shows no significant overall difference in the ability of these two functional types to place roots into nutrient-rich patches, but indicates other important differences among the two functional groups.     

Hydrogen peroxide is critical for UV-induced apoptosis inhibition

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O2*-) and hydrogen peroxide (H2O2), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H2O2-eliminating enzyme, catalase, but not in the presence of an O2*--eliminating enzyme, superoxide dismutase. Treatment with H2O2 itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H2O2 treatment decreased in H2O2-resistant cells. Although both UVB and H2O2 are known to induce DNA damage, other DNA damaging agents, like gamma-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H2O2 was essential to the inhibition of apoptosis, in which DNA damage had no role.     

Thaumatocotyle pseudodasybatis Hargis, 1955 (Monogenea: Monocotylidae) from Aetobatus cf. narinari, with a comparison of specimens from Australia, French Polynesia and New Caledonia

Thaumatocotyle pseudodasybatis Hargis, 1955, has previously been described from Aetobatus narinari in the Atlantic and subsequently recorded from the Pacific. Aetobatus cf. narinari is now considered a species complex; as monocotylids are often strictly species specific, we test the hypothesis that detailed examination of specimens of monocotylids from rays from various localities could reveal morphological differences and eventually help our understanding of the systematics of the host. T. pseudodasybatis, previously known from seven specimens only, is redescribed from an additional 26 specimens from the South Pacific (off New Caledonia, Australia and Ranguiroa, French Polynesia), all from Aetobatus cf. narinari. The female reproductive organs are described in detail. The distal extremity of the male sclerotised copulatory organ, described in detail for the first time, shows a characteristic pattern of longitudinal striations on its edge that might be useful for future distinction from other species. The development of the male and female organs in juveniles is described, showing that growth of the male sclerotised copulatory organ begins with its basal part and precedes development of the ejaculatory bulb. Specimens from New Caledonia, Australia and French Polynesia had similar measurements and morphology, especially in the shape of the distal end of the male sclerotised copulatory organ; they were also similar to the holotype from the Atlantic. This suggests that all specimens from the Pacific and Atlantic belong to a single species; T. pseudodasybatis thus cannot be used to differentiate populations of Aetobatus cf. narinari, perhaps because this monocotylid is not strictly species-specific.

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Résumé Thaumatocotyle pseudodasybatis Hargis, 1955 a été décrit d’Aetobatus narinari dans l’Atlantique, et plus tard retrouvé dans le Pacifique. Aetobatus cf. narinari est maintenant considéré comme un complexe d’espèces. Parce que les Monocotylidae sont souvent strictement spécifiques de leur espèce-hôte, nous testons l’hypothèse qu’un examen détaillé des spécimens de Monocotylidae de raies de localités variées pourrait révéler des différences morphologiques et finalement aider à comprendre la systématique de l’hôte. T. pseudodasybatis, connu précédemment par seulement sept spécimens, est redécrit à partir de 26 spécimens supplémentaires de l’Océan Pacifique Sud (Nouvelle-Calédonie, Australie, et Ranguiroa, Polynésie Française), tous de Aetobatus cf. narinari. Les organes reproducteurs femelles sont décrits en détail. L’extrémité distale de l’appareil copulateur sclérifié mâle, décrite en détail pour la première fois, montre un patron caractéristique de striations longitudinales sur son bord, qui pourrait être utile à l’avenir pour différencier cette espèce. Le développement des organes mâles et femelles chez les juvéniles est décrit; cela montre que la croissance de l’appareil copulateur sclérifié mâle commence par son extrémité basale et précède le développement du bulbe éjaculateur. Les spécimens de Nouvelle-Calédonie, Australie et Polynésie Française ont une morphologie et des mensurations similaires, en particulier pour la forme de l’extrémité distale de l’appareil copulateur sclérifié mâle. Ils sont aussi similaires à l’holotype, qui vient de l’Atlantique. Ceci suggère que tous les spécimens du Pacifique et de l’Atlantique appartiennent à une même espèce. T. pseudodasybatis ne peut donc pas être utilisé pour différencier les populations d’Aetobatus cf. narinari, peut-être parce que ce Monocotylidae n’est pas strictement spécifique.

     

Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation

The mechanisms of RecA-mediated three-strand homologous recombination are investigated at the single-molecule level, using magnetic tweezers. Probing the mechanical response of DNA molecules and nucleoprotein filaments in tension and in torsion allows a monitoring of the progression of the exchange in real time, both from the point of view of the RecA-bound single-stranded DNA and from that of the naked double-stranded DNA (dsDNA). We show that strand exchange is able to generate torsion even along a molecule with freely rotating ends. RecA readily depolymerizes during the reaction, a process presenting numerous advantages for the cell's 'protein economy' and for the management of topological constraints. Invasion of an untwisted dsDNA by a nucleoprotein filament leads to an exchanged duplex that remains topologically linked to the exchanged single strand, suggesting multiple initiations of strand exchange on the same molecule. Overall, our results seem to support several important assumptions of the monomer redistribution model.