Bone-targeted 2,6,9-trisubstituted purines: novel inhibitors of Src tyrosine kinase for the treatment of bone diseases

Novel bone-targeted 2,6,9-trisubstituted purine template-based inhibitors of Src tyrosine kinase are described. Drug design studies of known purine compounds revealed that both positions-2 and -6 were suitable for incorporating bone-seeking moieties. A variety of bone-targeting groups with different affinity to hydroxyapatite were utilized in the study. Compound 3d was determined to be a potent Src inhibitor and was quite selective against a panel of other protein kinases.     

Effect of methylation on the stability and solvation free energy of amylose and cellulose fragments: a molecular dynamics study

Molecular dynamics (MD) simulations were used to study the stability and solvation of amylose and cellulose fragments. The recently developed gromos carbohydrate force field was further tested by simulating maltose, cellobiose, and maltoheptaose. The MD simulations reproduced fairly well the favorable conformations of disaccharides defined by the torsional angles related with the glycosidic bond and the radius gyration of maltoheptaose. The effects of methylation at different hydroxyl groups on the stability of amylose and cellulose fragments were investigated. The methylations of O-2 and O-3 reduce the stability of a single helix more than methylation at O-6, while the latter reduces the stability of a double helix more. Solvation free-energy differences between the unsubstituted amylose and cellulose fragments and the methylated species were studied using the single-step perturbation method. It was found that methylation at O-2 has the biggest effect, in agreement with experiment.     

Postfusional control of quantal current shape

Whether glutamate is released rapidly, in an all-or-none manner, or more slowly, in a regulated manner, is a matter of debate. We analyzed the time course of excitatory postsynaptic currents (EPSCs) at glutamatergic neuromuscular junctions of Drosophila and found that the decay phase of EPSCs was protracted to a variable extent. The protraction was more pronounced in evoked and spontaneous quantal EPSCs than in action potential-evoked multiquantal EPSCs; reduced in quantal EPSCs from endophilin null mutants, which maintain release via kiss-and-run; and dependent on synaptotagmin isoform, calcium, and protein phosphorylation. Our data indicate that glutamate is released from individual synaptic vesicles for milliseconds through a fusion pore. Quantal glutamate discharge time course depends on presynaptic calcium inflow and the molecular composition of the release machinery.     

Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 x 10(3) to 4.4 x 10(9) copies ml(-1) or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 x 10(1) to 2.2 x10(6) copies ml(-1) or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml(-1). The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.     

A preliminary phylogeny of the Pterasteridae (Echinodermata,Asteroidea) and the first fossil record: Late Cretaceous of Germany and Belgium

The Pterasteridae comprises a diversified group of extant largely deep-sea starfishes. Generic diagnoses have been based classically on soft tissue characters and skeletal architecture. A preliminary phylogeny of sixteen extant species is here worked out by cladistic analysis. The resulting tree suggests monophyly of extant genera and the validity of dissociated plates for identification of genera. Fossil remains of Pterasteridae are here described for the first time. By comparison with extant species, all the skeletal remains from the lower Upper Campanian of Belgium and from the lower Maastrichtian of Germany are tentatively assigned to the genusPteraster. The fossil record of starfishes is poor, but the present Late Cretaceous pterasterids provide one more piece of evidence of the high diversity of starfishes during the Mesozoic. Known Late Cretaceous and Paleogene fossils are broadly similar, which suggests the end-Cretaceous extinction event did not cause major turnover in asteroid faunal composition. As suggested for other starfish groups, both the fossil record of deep-sea Pterasteridae in shelf settings and tree topology imply an onshore-offshore evolutionary trend.      

Simultaneous determination of ketamine and xylazine in canine plasma by liquid chromatography with ultraviolet absorbance detection

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of ketamine and xylazine in canine plasma is described. Plasma samples (500 microl) are cleaned up via liquid-liquid extraction. The analytes and the internal standard clonidine are separated on a cyano (CN) column using a mobile phase containing acetonitrile-0.005 M phosphate buffer adjusted to pH 5.5 (3:2) at a detection wavelength of 215 nm. The method was validated according to specificity, sensitivity, accuracy and reproducibility and was used to determine the plasma concentrations of both compounds in dogs after intramuscular injection.