Differences in regulation of the first two M-phases in Xenopus laevis embryo cell-free extracts

The first embryonic M-phase is special, being the time when paternal and maternal chromosomes mix together for the first time. Reports from a variety of species suggest that the regulation of first M-phase has many particularities; however, no systematic comparative study of the biochemical aspects of first and the following M-phases has been previously undertaken. Here, we ask whether the regulation of the first embryonic M-phase is modified, using Xenopus cell-free extracts. We developed new types of extract specific for the first and the second M-phase obtained either from parthenogenetic or from in vitro fertilized embryos. Analyses of these extracts confirmed that the amplitude of histone H1 kinase activity reflecting CDK1/cyclin B (or MPF for M-phase Promoting Factor) activity is higher and persists longer than during the second M-phase, and that levels of cyclins B1 and B2 are correspondingly higher during the first than the second embryonic M-phase. Inhibition of protein synthesis shortly before M-phase entry reduced mitotic histone H1 kinase amplitude, shortened the period of mitotic phosphorylation of chosen marker proteins, and reduced cyclin B1 and B2 levels, suggesting a role of B-type cyclins in regulating the duration of mitotic events. Moreover, addition of exogenous cyclin B to the extract prior the second mitosis brought forward the activation of mitotic histone H1 kinase but prolonged the duration of this activity. We also confirmed that the inhibitory phosphorylation of CDK1 on tyrosine 15 oscillates between the first two embryonic M-phases, but is clearly more pronounced before the first than the second mitosis, while the MAP kinase ERK2 tended to show greater activation during the first embryonic M-phase but with a similar duration of activation. We conclude that discrete differences exist between the first two M-phases in Xenopus embryo and that higher CDK1/cyclin B activity and B-type cyclin levels could account for the different characteristics of these M-phases.     

The Ry-fsto gene from Solanum stoloniferum for extreme resistant to Potato virus Y maps to potato chromosome XII and is diagnosed by PCR marker GP122718 in PVY resistant potato cultivars

A novel locus for extreme resistance to Potato virus Y (PVY), Ry-f_sto, was identified on potato chromosome XII. The gene Ry-f_sto has been introgressed from the wild potato species Solanum stoloniferum. Inheritance of Ry-f_sto in the tetraploid potato population Rysto was consistent with the model of a single, dominant gene. Bulked segregant analysis identified an ISSR (inter-simple sequence repeat) marker UBC 857₉₈₀ linked to Ry-f_sto. This marker mapped to linkage group XII of a reference potato RFLP (restriction fragment length polymorphism) map. Chromosome XII specific RFLP markers were converted into PCR-based STS and CAPS markers and tested for linkage with Ry-f_sto in the population Rysto. CAPS marker GP122₇₁₈ was tightly linked to the resistance gene and was successfully used to identify Polish and German cultivars expressing extreme resistance to PVY. This indicates that the source of Ry-f_sto has been widely utilized in various potato breeding programs and can be monitored by a diagnostic marker in marker-assisted selection.     

Novel dinucleoside 5',5'-triphosphate cap analogues. Synthesis and affinity for murine translation factor eIF4E

Chemical synthesis of a series of novel dinucleoside cap analogues, m7GpppN, where N is formycin A, 3'-O-methylguanosine, 9-beta-D-arabinofuranosyladenine, and isoguanosine, has been performed using our new methodology. The key reactions of pyrophosphate bonds formation were achieved in anhydrous dimethylformamide solutions employing the catalytic properties of zinc salts. Structures of the new cap analogues were confirmed by 1H NMR and 31p NMR spectra. The binding affinity of the new cap analogues for murine eIF4E(28-217) were determined spectroscopically showing the highest association constant for the analogue that contains formycin A.     

Oxidative transformations of nucleoside fluorenemethyl H-phosphonoselenoate diesters

9-Fluorenemethyl H-phosphonoselenoate monoester has been used to produce thymidine 3'-O-phosphoroselenoate monoester from which various P(V) derivatives containing multiple modifications at phosphorus were obtained; e.g., thymidine 3'-O-phosphoroselenofluoridate, 3'-O-phosphoroselenothioate, or 3'-O-phosphorodiselenoate monoesters.     

From cluster calculations to molecular materials: a mixed pseudopotential approach to modeling mixed-valence systems

In this paper we present a technique for finding an appropriate parameterization of ultrasoft pseudopotentials for modeling mixed-valence materials. For the example of hexacyanometallate molecular building blocks, we show how ionic cluster calculations can be used to determine a set of parameters for the metal centers. Pseudopotentials chosen in such a way are then shown to be suitable for periodic calculations of the corresponding mixed-valence materials (e.g., Prussian Blue).This work was originally presented at the Modelling and Design of Molecular Materials conference in Wrocaw, Poland.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.