Century-scale Methylome Stability in a Recently Diverged Arabidopsis thaliana Lineage

There has been much excitement about the possibility that exposure to specific environments can induce an ecological memory in the form of whole-sale, genome-wide epigenetic changes that are maintained over many generations. In the model plant Arabidopsis thaliana, numerous heritable DNA methylation differences have been identified in greenhouse-grown isogenic lines, but it remains unknown how natural, highly variable environments affect the rate and spectrum of such changes. Here we present detailed methylome analyses in a geographically dispersed A. thaliana population that constitutes a collection of near-isogenic lines, diverged for at least a century from a common ancestor. Methylome variation largely reflected genetic distance, and was in many aspects similar to that of lines raised in uniform conditions. Thus, even when plants are grown in varying and diverse natural sites, genome-wide epigenetic variation accumulates mostly in a clock-like manner, and epigenetic divergence thus parallels the pattern of genome-wide DNA sequence divergence.     

Wolbachia distribution in selected beetle taxa characterized by PCR screens and MLST data

Wolbachia (Alphaproteobacteria) is an inherited endosymbiont of arthropods and filarial nematodes and was reported to be widespread across insect taxa. While Wolbachia's effects on host biology are not understood from most of these hosts, known Wolbachia‐induced phenotypes cover a spectrum from obligate beneficial mutualism to reproductive manipulations and pathogenicity. Interestingly, data on Wolbachia within the most species‐rich order of arthropods, the Coleoptera (beetles), are scarce. Therefore, we screened 128 species from seven beetle families (Buprestidae, Hydraenidae, Dytiscidae, Hydrophilidae, Gyrinidae, Haliplidae, and Noteridae) for the presence of Wolbachia. Our data show that, contrary to previous estimations, Wolbachia frequencies in beetles (31% overall) are comparable to the ones in other insects. In addition, we used Wolbachia MLST data and host phylogeny to explore the evolutionary history of Wolbachia strains from Hydraenidae, an aquatic lineage of beetles. Our data suggest that Wolbachia from Hydraenidae might be largely host genus specific and that Wolbachia strain phylogeny is not independent to that of its hosts. As this contrasts with most terrestrial Wolbachia–arthropod systems, one potential conclusion is that aquatic lifestyle of hosts may result in Wolbachia distribution patterns distinct from those of terrestrial hosts. Our data thus provide both insights into Wolbachia distribution among beetles in general and a first glimpse of Wolbachia distribution patterns among aquatic host lineages.     

Sequence elements in both subunits of the DNA fragmentation factor are essential for its nuclear transport

DNA cleavage is a biochemical hallmark of apoptosis. In humans, apoptotic DNA cleavage is executed by DNA fragmentation factor (DFF) 40. In proliferating cells DFF40 is expressed in the presence of its chaperone and inhibitor DFF45, which results in the formation of the DFF complex. Here, we present a systematic analysis of the nuclear import of the DFF complex. Our in vitro experiments demonstrate that the importin alpha/beta-heterodimer mediates the translocation of the DFF complex from the cytoplasm to the nucleus. Both DFF subunits interact directly with the importin alpha/beta-heterodimer. However, importin alpha/beta binds more tightly to the DFF complex compared with the individual subunits. Additionally, the isolated C-terminal regions of both DFF subunits together bind importin alpha/beta more strongly than the individual C termini. Our results from in vivo studies reveal that the C-terminal regions of both DFF subunits harbor nuclear localization signals. Furthermore, nuclear import of the DFF complex requires the C-terminal regions of both subunits. In more detail, one basic cluster in the C-terminal region of each subunit, DFF40 (RLKRK) and DFF45 (KRAR), is essential for nuclear accumulation of the DFF complex. Based on these findings two alternative models for the interaction of importin alpha/beta with the DFF complex are presented.     

cGMP signals mainly through cAMP kinase in permeabilized murine aorta

GMP affects vascular tone by multiple mechanisms, including inhibition of the Rho/Rho kinase-mediated Ca(2+) sensitization, a process identified as Ca(2+) desensitization. Ca(2+) desensitization is mediated probably by both cGMP- and cAMP-dependent protein kinases (cGKI and PKA). We investigate to which extent Ca(2+) desensitization is initiated by cGKI and PKA. cGMP/cAMP-induced relaxation was studied at constant [Ca(2+)] in permeabilized aortas from wild-type and cGKI-deficient mice. [Ca(2+)] increased aortic tone in the absence and presence of 50 microM GTPgammaS with EC(50) values of 160 and 30 nM, respectively. In the absence of GTPgammaS, the EC(50) for [Ca(2+)] was shifted rightward from 0.16 microM to 0.43 and 0.82 microM by 1 and 300 microM 8-bromo-cGMP (8-Br-cGMP), and to 8 microM by 10 microM Y-27632. Contractions induced by 300 nM [Ca(2+)] were relaxed by 8-Br-cGMP with an EC(50) of 2.6 microM. Surprisingly, [Ca(2+)]-induced contractions were also relaxed by 8-Br-cGMP in aortas from cGKI(-/-) mice (EC(50) of 19 microM). Western blot analysis of the vasodilator-stimulated phosphoprotein indicated "cross"-activation of PKA by 1 mM 8-Br-cGMP in aortic smooth muscle cells from cGKI(-/-) mice. Indeed, the PKA inhibitor peptide (PKI 5-24) completely abolished the relaxant effect of 8-Br-cGMP in muscles from cGKI(-/-) mice and to 65% in wild-type aortas. The thromboxane analogue U-46619 induced contraction at constant [Ca(2+)], which was only partially relaxed by 8-Br-cGMP but completely relaxed by Y-27632. The effect of 8-Br-cGMP on U-46619-induced contraction was attenuated by PKI 5-24. These results show that cGKI has only a small inhibitory effect on Ca(2+) sensitization in murine aortas.     

Arabidopsis: a model genus for speciation

What genetic and epigenetic changes underlie adaptation and divergence? Arabidopsis thaliana and its relatives are increasingly being employed to address such central questions of evolutionary biology. For example, comparative, genomic and classical genetic approaches are revealing mechanisms underlying processes relevant to speciation, including mating system evolution, the effects of ploidy and other chromosomal differences, and the roles that specific genes might play in Dobzhansky-Muller type incompatibilities. The considerable body of knowledge and resources available for A. thaliana and improvements in tools and technology applied to its close relatives are opening doors for combining experimental and comparative analyses to elucidate fundamental mechanisms of evolution.     

Synthesis, liposomal preparation, and in vitro toxicity of two novel dodecaborate cluster lipids for boron neutron capture therapy

A new class of lipids, containing the closo-dodecaborate cluster, has been synthesized. Two lipids, S-(N, N-(2-dimyristoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-14) and S-(N, N-(2-dipalmitoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-16) are described. Both of them have a double-tailed lipophilic part and a headgroup carrying two negative charges. Differential scanning calorimetry shows that B-6-14 and B-6-16 bilayers have main phase transition temperatures of 18.8 and 37.9 degrees C, respectively. Above the transition temperature of 18.8 degrees C, B-6-14 can form liposomal vesicles, representing the first boron-containing lipid with this capability. Upon cooling below the transition temperature, stiff bilayers are formed. When incorporated into liposomal formulations with equimolar amounts of distearoyl phosphatidylcholine (DSPC) and cholesterol, stable liposomes are obtained. The zeta-potential measurements indicate that both B-6-14- and B-6-16-containing vesicles are negatively charged, with the most negative potential described of any liposome so far. The liposomes are of high potential value as transporters of boron to tumor cells in treatments based on boron neutron capture therapy (BNCT). Liposomes prepared from B-6-14 were slightly less toxic in V79 Chinese hamster cells (IC50 5.6 mM) than unformulated Na2B12H11SH (IC50 3.9 mM), while liposomes prepared from B-6-16 were not toxic even at 30 mM.     

Molecular phylogeny of the Heterotrichea (Ciliophora, Postciliodesmatophora) based on small subunit rRNA gene sequences

A comprehensive molecular analysis of the phylogenetic relationships within the Heterotrichea including all described families is still lacking. For this reason, the complete nuclear small subunit (SSU) rDNA was sequenced from further representatives of the Blepharismidae and the Stentoridae. In addition, the SSU rDNA of a new, undescribed species of the genus Condylostomides (Condylostomatidae) was sequenced. The detailed phylogenetic analyses revealed a consistent branching pattern: while the terminal branches are generally well resolved, the basal relationships remain unsolved. Moreover, the data allow some conclusions about the macronuclear evolution within the genera Blepharisma, Stentor, and Spirostomum suggesting that a single, compact macronucleus represents the ancestral state.