Use of mitochondrial and nuclear genes to infer the origin of two endemic pigeons from the Canary Islands

DNA nucleotide sequences from two mitochondrial genes (cytochrome b and NADH dehydrogenase subunit 2) and the nuclear intron 7 of β-fibrinogen were obtained to infer the phylogenetic origin of the two endemic Canarian pigeons: Bolle’s Pigeon (Columba bollii) and Laurel Pigeon (C. junoniae). Phylogenetic analyses of mitochondrial and nuclear genes based on maximum parsimony, maximum likelihood and Bayesian inference all converged into a congruent topology: C. bollii clusters together with the Wood Pigeon (C. palumbus) which is common in Europe and Asia, while C. junoniae was found near the base of the clade that includes other species of the genus Columba from the Old World. Laurel Pigeon probably represents an old lineage that might have colonized the Canary Islands a long time ago (20 My) while Bolle’s Pigeon might have arrived on the archipelago much later during the Upper Miocene (5 My). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities

The coding sequence, which corresponds to the mature antimicrobial peptide ranalexin from the frog Rana catesbeiana, was chemically synthesized with preferred codons for expression in Escherichia coli. It was cloned into the vector pET32c (+) to express a thioredoxin-ranalexin fusion protein which was produced in soluble form in E. coli BL21 (DE3) induced under optimized conditions. After two purification steps through affinity chromatography, about 1 mg of the recombinant ranalexin was obtained from 1 L of culture. Mass spectrometrical analysis of the purified recombinant ranalexin demonstrated its identity with ranalexin. The purified recombinant ranalexin is biologically active. It showed antibacterial activities similar to those of the native peptide against Staphylococcus aureus, Streptococcus pyogenes, E. coli, and multidrug-resistant strains of S. aureus with minimum inhibitory concentration values between 8 and 128 μg/ml. The recombinant ranalexin is also cytotoxic in HeLa and COS7 human cancer cells (IC₅₀?=?13–15 μg/ml).     

Corynebacterium heidelbergense sp. nov., isolated from the preen glands of Egyptian geese (Alopochen aegyptiacus)

Two strains (pedersoli^T and girotti) of a new species of bacteria were isolated from the preen glands of wild Egyptian geese (Alopochen aegyptiacus) from the river Neckar in southern Germany in two subsequent years. The strains were lipophilic, fastidious, Gram-positive rods and belonged to the genus Corynebacterium. Phylogenetically, the isolates were most closely related to Corynebacterium falsenii DSM 44353^T which has been found to be associated with birds before. 16S rRNA gene sequence similarity to all known Corynebacterium spp. was significantly <97%. Corresponding values of rpoB showed low levels of similarity <87% and ANIb was <73%. G + C content of the genomic DNA was 65.0 mol% for the type strain of the goose isolates, as opposed to 63.2 mol% in Corynebacterium falsenii. MALDI-TOF MS analysis of the whole-cell proteins revealed patterns clearly different from the related species, as did biochemical tests, and polar lipid profiles. We therefore conclude that the avian isolates constitute strains of a new species, for which the name Corynebacterium heidelbergense sp. nov. is proposed. The type strain is pedersoli^T (=DSM 104638^T = LMG 30044^T).     

Organization and function of an actin cytoskeleton in Plasmodium falciparum gametocytes

In preparation for transmission to its mosquito vector, Plasmodium falciparum, the most virulent of the human malaria parasites, adopts an unusual elongated shape. Here we describe a previously unrecognized actin‐based cytoskeleton that is assembled in maturing P. falciparum gametocytes. Differential extraction reveals the presence of a highly stabilized population of F‐actin at all stages of development. Super‐resolution microscopy reveals an F‐actin cytoskeleton that is concentrated at the ends of the elongating gametocyte but extends inward along the microtubule cytoskeleton. Formin‐1 is also concentrated at the gametocyte ends suggesting a role in actin stabilization. Immunoelectron microscopy confirms that the actin cytoskeleton is located under the inner membrane complex rather than in the sub‐alveolar space. In stage V gametocytes, the actin and microtubule cytoskeletons are reorganized in a coordinated fashion. The actin‐depolymerizing agent, cytochalasin D, depletes actin from the end of the gametocytes, whereas the actin‐stabilizing compound, jasplakinolide, induces formation of large bundles and prevents late‐stage disassembly of the actin cytoskeleton. Long‐term treatment with these compounds is associated with disruption of the normal mitochondrial organization and decreased gametocyte viability.     

Mitochondrial phylogeography of the European pond turtle, Emys orbicularis (Linnaeus 1758)

The phylogeny and phylogeography of Emys orbicularis was inferred from mitochondrial nucleotide sequences of the cytochrome b gene analysed by DNA sequencing and RNA heteroduplex analysis. Within the family Emydidae the monotypic genus Emys is affiliated with the nearctic taxa Emydoidea blandingii and Clemmys marmorata. The analysis of 423 individuals of E. orbicularis, originating throughout its distribution range, revealed a remarkable intraspecific differentiation in 20 different haplotypes with distinct geographical ranges. Maximum parsimony analysis produced a star-like phylogeny with seven main lineages which may reflect separations in the late Pliocene. The haplotype distribution examined by partial Mantel tests and analysis of molecular variance revealed a substantial effect of glacial periods. This historical perspective suggests the existence of multiple glacial refugia and considerable Holocene range expansion which was modulated by climatic traits. Further support is gained for the occurrence of long-term parapatry in glacial refugia.     

Preference–performance relationship and influence of plant relatedness on host use by Pityogenes chalcographus L.

• 1 Pityogenes chalcographus L. (Coleoptera: Scolytinae) causes damage in European coniferous forests, primarily on Picea abies L. Karst., but is also recorded on other native and exotic Pinaceae species. Estimating the adequacy between adult preference and larval performance of this beetle among its host‐range, as well as the influence of plant taxonomic relatedness on these parameters, would provide useful information on the beetle's ability to shift onto novel hosts.
• 2 Choice and no‐choice assays were conducted under laboratory conditions. Adult preference and larval performance parameters among two native (Pinus sylvestris L. and Picea abies) and three exotic north American [Pinus contorta Dougl., Picea sitchensis (Bong.) Carr. and Pseudotsuga menziesii Mirbel (Franco)] conifer species were measured.
• 3 Pityogenes chalcographus exhibited a significant positive relationship between preference and performance. Picea abies was both the preferred and the most suitable host species for larval development. The closest relative, P. sitchensis, was the second best choice in terms of preference and performance. Pseudotsuga menziesii occupied an intermediate position for both beetle preference and performance, and Pinus spp. were the least suitable hosts for beetle development.
• 4 Adult preference and larval performance ranking among hosts provides little support to the plant taxonomic relatedness hypothesis. Taxonomic relatedness could play a role on the diet breadth, although only at a limited scale, within the genus Picea. At higher taxonomic levels, other factors such as bark thickness might be decisive.

     

Phylogeography and population structure of the saker falcon (Falco cherrug) and the influence of hybridization: mitochondrial and microsatellite data

Microsatellite as well as sequence analysis of the mitochondrial control region were applied to infer phylogeography and population genetic structure of the saker falcon (Falco cherrug). Furthermore, we compared the patterns of mitochondrial haplotypes with the variation of microsatellite alleles among the species of the hierofalcon complex (F. cherrug, Falco rusticolus, Falco biarmicus, Falco jugger) to test hypotheses on population history. Historical samples from museum specimens of F. cherrug were analysed together with samples from contemporary populations to investigate possible influences of hybrid falcons escaped from falconry on the genetic composition. In the mitochondrial DNA analysis, none of the four species represents a monophyletic group. Moreover, there are no clearly defined groups of haplotypes corresponding to taxonomic entities. In the microsatellite analysis most of the variation is shared between species and no clear differentiation by private alleles is found. Yet, with a Bayesian clustering method based on allele frequencies, a differentiation of F. cherrug, F. rusticolus and two geographic groups of F. biarmicus was detected. Results from both nuclear and mitochondrial markers are compatible with the previously postulated 'Out of Africa' hypothesis assuming an African origin of the hierofalcons. From an ancestral African population, F. cherrug, F. rusticolus and F. jugger split off in separate waves of immigration into Eurasia and South Asia. A combination of evolutionary processes, including incomplete lineage sorting as well as hybridization, may be responsible for the currently observed genetic patterns in hierofalcons.     

N-Methylation of quinolizidine alkaloids: an S-adenosyl-l-methionine: cytisine N-methyltransferase from Laburnum anagyroides plants and cell cultures of L. alpinum and Cytisus canariensis

An S-adenosyl-l-methionine (SAM): cytisine N-methyltransferase could be demonstrated in crude enzyme preparations from Laburnum anagyroides plants and cell cultures of L. alpinum and Cytisus canariensis. The transferase specifically catalyzes the transfer of a methyl group from SAM to cytisine. The apparent K_m values are 60 mol l^-1 for cytisine and 17 mol l^-1 for SAM. Other quinolizidine alkaloids, e.g. angustifoline and albine, are N-methylated by only 10–15%. The transferase shows a pH optimum at pH 8.5. It is activated by dithioerythritol and inhibited by thiol reagents and Fe²⁺ and Fe³⁺. The reaction product S-adenosylhomocysteine is a powerful inhibitor of the transferase reaction. Cell cultures of L. alpinum which have an active SAM: cytisine N-methyltransferase and which are able to N-methylate exogenous cytisine in vivo, do not accumulate cytisine or N-methylcytisine to a detectable degree.Abbreviations GLC gas-liquid chromatography - SAM S-adenosylmethionine - TLC thin-layer chromatography     

Purification of plasma membranes of rat mammary gland : Comparisons of subfractions with rat milk fat globule membrane

Partially purified plasma membranes of rat mammary gland, obtained as light (F₁) and heavy (F₂) fractions by flotation on a discontinuous sucrose density gradient, were further fractionated by density perturbation flotation using digitonin to shift the density of the cholesterol-rich portion of the membranes. The shifted fraction (F₁F₃) of digitonin-treated F₁ was highly enriched in 5′-nucleotidase, cholesterol and sialic acid, but free of galactosyltransferase, suggesting that it contained highly purified plasma membranes. The unshifted fraction (F₁DF₁) was enriched in galactosyltransferase and depleted in nucleotidase, cholesterol and sialic acid, suggesting that it contained Golgi fragments. The F₂ fraction shows substantially different behavior. Part of it re-equilibrates to the F₁ position upon reflotation. When treated with digitonin, part of F₂ is shifted to a higher density (F₂DF₃). F₂DF₃ is enriched in 5′-nucleotidase, cholesterol, sialic acid and galactosyltransferase. These properties suggest that this subfraction comes from a plasma membrane containing galactosyltransferase.The sialoglycoproteins of the various fractions were compared with those of rat milk fat globule membrane, which is derived in part from the apical surface of the mammary secretory cell. Dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals two major glycoprotein bands (GP-II and GP-III) in F₁DF₃. F₂DF₃ contains these and an additional band of lower mobility (GP-I). Both crude and purified MFGM contain all three bands. Comparisons of peanut lectin receptors by autoradiography of polyacrylamide gels run in SDS and then treated with [¹²⁵I]peanut lectin also suggest that F₂DF₃ is more similar to the milk fat globule membrane than is F₁DF₃. However, analysis of the membrane polypeptides and concanavalin A (ConA) receptors shows no obvious relationship between milk fat globule membrane and any of the isolated mammary membrane fractions. These results indicate that the relationship between the milk fat globule membrane and mammary membranes is complex, possibly involving components not associated with the mammary plasma membrane or only selected components of the plasma membrane.