Molecular evidence for the presence of cryptic evolutionary lineages in the freshwater copepod genus <Emphasis Type="Italic">Hemidiaptomus</Emphasis> G.O. Sars, 1903 (Calanoida,Diaptomidae)

The pattern of morphological and mtDNA cytochrome b diversity of three calanoid copepod species belonging to the diaptomid genus Hemidiaptomus has been investigated with the aim of checking the reliability of the morphological characters currently used for species identification, and the possible presence of cryptic taxa. A sharply different molecular structuring has been observed in the studied species: while Hemidiaptomus amblyodon exhibits a remarkable constancy throughout the European range of its distribution area (maximum inter-populations cytochrome b divergence of 3%), observed distances between presumed conspecific lineages of Hemidiaptomus gurneyi (maximum divergence of 21.5%) and Hemidiaptomus ingens (maximum 19.1%) suggest that under these binomens are in fact included complexes of cryptic, or currently just unrecognized, independent evolutionary lineages. The application of the “4x rule” shows that the two lineages singled out within H. ingens are in fact independent evolutionary units, while the complex molecular structure observed in H. gurneyi s.l. could not be resolved based on the currently available data. Applying standard crustacean mtDNA evolutionary rates to the observed divergence values, the separation of the main lineages within both H. ingens and H. gurneyi might dates back to the Miocene; however, it has also to be considered that the rate of mtDNA evolution might be accelerated in copepods, as already observed in other arthropod taxa. Present results gives further evidences of the high potential for copepod speciation with no or little morphological changes, and stress the need of a revision of the most controversial Palaearctic diaptomid genera.     

Pleistocene diversification in Morocco and recent demographic expansion in the Mediterranean pond turtle Mauremys leprosa

Quaternary climatic oscillations and geographic barriers have strongly influenced the distribution and diversification of thermophilic species occurring in the Mediterranean Basin. The Western Mediterranean pond turtle, Mauremys leprosa, is widely distributed throughout the Iberian Peninsula, southern France and most of the Maghreb region, with two subspecies currently recognized. In this work, we used 566 samples, including 259 new individuals, across the species range, and sequenced two mitochondrial markers (cytochrome b gene and control region; 163 samples in a concatenated mtDNA dataset) and one nuclear intron (R35; 23 samples representing all identified sublineages) to study the evolutionary history of M. leprosa. We combined phylogenetic methods and phylogeographic continuous diffusion models with spatial analysis. Our results (1) show a high level of genetic structure in Morocco originated during the Pleistocene; (2) reveal two independent population expansion waves from Morocco to Tunisia and to southern Europe, which later expanded throughout the Iberian Peninsula, and (3) identify several secondary contact zones in Morocco. Our study also sheds new light on the role of geographical features (Moroccan mountains ranges and the Strait of Gibraltar) and Pleistocene climatic oscillations in shaping genetic diversity and structure of M. leprosa, and underlines the importance of the Maghreb as a differentiation centre harbouring distinct glacial refugia.     

Specificity of Lysine:N-Hydroxylase: A Hypothesis for a Reactive Substrate Intermediate in the Catalytic Mechanism

The recombinant cytoplasmic preparation of lysine:N⁶-hydroxlase catalyzes the conversion ofL-lysine to itsN⁶-hydroxy derivative when supplemented with the cofactors NADPH and FAD. A number of lysine analogs reflecting minor alterations in the inherent structural features of the amino acid as well compounds with relatively high affinity for lysine binding domains in other proteins were examined for their ability to serve as substrates of lysine:N⁶-hydroxylase. These studies have revealed that the enzyme does not tolerate any change in the structural features ofL-lysine, its preferred substrate, with the exception of the replacement of the C_γH₂-methylene group by sulfur, as in (S)-2-aminoethyl-L-cysteine.L-Norleucine is a potent inhibitor of the enzyme whileL-norvaline andL-α-aminobutyric acid do not exhibit such effect, indicating the importance of a C₄hydrophobic side chain for effective interaction with the enzyme. Among theN-alkyl amides of hydrophobic amino acids, onlyL-norleucine methylamide andL-α-aminobutyric acid ethylamide serve as moderate inhibitors of lysine:N⁶-hydroxylase. Based on the enzyme's stringent substrate specificity, a mechanism involving the conversion ofL-lysine to 2-aminocaprolactam prior to its oxygenation by the 4a-peroxyflavin intermediate in the catalytic cycle is proposed.     

Contrasting dynamics of water and mineral nutrients in stems shown by stable isotope tracers and cryo‐SIMS

Lateral exchange of water and nutrients between xylem and surrounding tissues helps to de‐couple uptake from utilization in all parts of a plant. We studied the dynamics of these exchanges, using stable isotope tracers for water (H₂¹⁸O), magnesium (²⁶Mg), potassium (⁴¹K) and calcium (⁴⁴Ca) delivered via a cut stem for various periods to the transpiration stream of bean shoots (Phaseolus vulgaris cv. Fardenlosa Shiny). Tracers were subsequently mapped in stem cross‐sections with cryo‐secondary ion mass spectrometry. The water tracer equilibrated within minutes across the entire cross‐section. In contrast, the nutrient tracers showed a very heterogeneous exchange between xylem vessels and the different stem tissues, even after 4 h. Dynamics of nutrients in the tissues revealed a fast and extensive exchange of nutrients in the xylem parenchyma, with, for example, calcium being completely replaced by tracer in less than 5 min. Dilution of potassium tracer during its 30 s transit in xylem sap through the stem showed that potassium concentration was up‐regulated over many hours, to the extent that some of it was probably supplied by phloem recirculation from the shoot.     

DNA taxonomy of the potamid freshwater crabs from Northern Africa (Decapoda,Potamidae)

The Mediterranean area, from southern Balkans to western Maghreb, is inhabited by the Potamon subgenus Euthelphusa, with three currently recognised species. The Maghrebian species P. (E.) algeriense is isolated from other Potamon species by the Mediterranean Sea and the Sahara Desert, and nearly no molecular data are currently available for this taxon. Present study investigated the mtDNA and nuDNA diversity in Potamon algeriense s.l. with the aims of exploring its molecular diversity pattern throughout its known distribution range, and testing the possible presence of cryptic taxa currently lumped under this binomen. The phylogenetic and DNA taxonomy analyses showed the presence of two well-supported clades of species rank within P. algeriense s.l, with an eastern clade including the populations from Tunisia and Numidia, and a western, highly structured clade including the populations from central Algeria and Morocco. In agreement with a typical Maghrebian biogeographic pattern, the distribution of these two species shows a clear east–west divide, with a disjunction zone located in Kabylia, and a strong link between molecular diversity and segregation within different hydrographical basins is evident. The Maghreb thus proved to host an unexpectedly high genetic diversity of, and to constitute a biodiversity hot-spot for, the Potamon subgenus Euthelphusa. In the light of the existence of two well-characterised species currently lumped under P. algeriense s.l., and of their noteworthy molecular structuring, the status of Maghrebian Potamon populations should be re-assessed for both the species present in the area, which are to be considered as independent management units.     

Different lineages of freshwater jellyfishes (Cnidaria,Olindiidae, Craspedacusta) invading Europe: another piece of the puzzle from Sicily,Italy

Limnology - Correctly identifying and mapping the distribution patterns of passively dispersing freshwater organisms is an important task to understand the mechanisms through which these organisms...     

The role of tryptophan residues in substrate binding to catalytic domains A and B of xylanase C from Fibrobacter succinogenes S85

Oxidation of the isolated catalytic domain B of xylanase C (XynC-B) from Fibrobacter succinogenes with N-bromosuccinimide (NBS) resulted in the modification of five of the seven Trp residues present in the enzyme. Hydrolytic activity of the enzyme was rapidly lost upon initiation of oxidation as a molar ratio of about two NBS molecules per molar equivalent of protein was sufficient to cause 50% inhibition of enzyme activity, and the addition of five molar equivalents of NBS resulted in less than 10% activity. Pre-incubation of XynC-B with the competitive inhibitor D-xylose resulted in the apparent protection of two Trp residues from oxidation. Xylose protection of the enzyme also resulted in a maintenance of activity, with 60% activity still evident after addition of 8-9 molar equivalents of NBS. This protection from inactivation was enhanced by the inclusion of xylohexaose in reaction mixtures. Under these conditions, however, a further Trp residue was protected from NBS oxidation. The three protected Trp residues were identified as Trp135, Trp161 and Trp202 by differential labelling and peptide mapping of NBS-oxidized preparations of the xylanase employing a combination of electrospray mass spectroscopic analysis and N-terminal sequencing. By analogy to the known structures of the family 11 xylanases, the fully conserved Trp202 residue is located on the only alpha-helix present in the enzymes, at the interface between it and the back of the beta-sheet which forms the active site cleft. Trp135 represents a highly conserved aromatic residue in family 11, but it is replaced with Thr in domain A of F. succinogenes xylanase C. To investigate the role of Trp135 in conferring the different activity profile of domain B relative to domain A, the Trp135Thr and Trp135Ala derivatives of domain B were prepared by site-directed mutagenesis. However, the kinetic parameters of the two domain B derivatives were not significantly different compared to the wild-type enzyme as reflected by K(M) and k(cat) values and product distribution profiles. Similar results were obtained with the Trp161Ala derivative of domain B, indicating that these two residues do not directly participate in the binding of substrate but likely form the foundation for binding subsite 2.     

Investigation of the molecular similarity in closely related protein systems: The PrP case study

The amyloid conversion is a massive detrimental modification affecting several proteins upon specific physical or chemical stimuli characterizing a plethora of diseases. In many cases, the amyloidogenic stimuli induce specific structural features to the protein conferring the propensity to misfold and form amyloid deposits. The investigation of mutants, structurally similar to their native isoform but inherently prone to amyloid conversion, may be a viable strategy to elucidate the structural features connected with amyloidogenesis. In this article, we present a computational protocol based on the combination of molecular dynamics (MD) and grid‐based approaches suited for the pairwise comparison of closely related protein structures. This method was applied on the cellular prion protein (PrP^C) as a case study and, in particular, addressed to the quali/quantification of the structural features conferred by either E200K mutations and treatment with CaCl₂, both able to induce the scrapie conversion of PrP. Several schemes of comparison were developed and applied to this case study, and made up suitable of application to other protein systems. At this purpose an in‐house python codes has been implemented that, together with the parallelization of the GRID force fields program, will spread the applicability of the proposed computational procedure. Proteins 2015; 83:1751–1765. © 2015 Wiley Periodicals, Inc.