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Poster presentation at 5th German Conference on Cheminformatics: 23. CIC-Workshop Goslar, Germany. 8-10 November 2009 Protein kinases are important targets for drug development. The almost identical protein folding of kinases and the common co-substrate ATP leads to the problem of inhibitor selectivity. Type II inhibitors, targeting the inactive conformation of kinases, occupy a hydrophobic pocket with less conserved surrounding amino acids. Human polo-like kinase 1 (Plk1) represents a promising target for approaches to identify new therapeutic agents. Plk1 belongs to a family of highly conserved serine/threonine kinases, and is a key player in mitosis, where it modulates the spindle checkpoint at metaphase/anaphase transition. Plk1 is over-expressed in all today analyzed human tumors of different origin and serves as a negative prognostic marker in cancer patients. The newly identified inhibitor, SBE13, a vanillin derivative, targets Plk1 in its inactive conformation. This leads to selectivity within the Plk family and towards Aurora A. This selectivity can be explained by docking studies of SBE13 into the binding pocket of homology models of Plk1, Plk2 and Plk3 in their inactive conformation. SBE13 showed anti-proliferative effects in cancer cell lines of different origins with EC50 values between 5 microM and 39 microM and induced apoptosis. Increasing concentrations of SBE13 result in increasing amounts of cells in G2/M phase 13 hours after double thymidin block of HeLa cells. The kinase activity of Plk1 was inhibited with an IC50 of 200 pM. Taken together, we could show that carefully designed structure-based virtual screening is well-suited to identify selective type II kinase inhibitors targeting Plk1 as potential anti-cancer therapeutics.
Background: Phototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea. Methodology/Principal Findings: We developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis. Conclusion/Significance: This is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis.
Recently a first genome-wide analysis of translational regulation using prokaryotic species had been performed which revealed that regulation of translational efficiency plays an important role in haloarchaea. In fact, the fractions of genes under differential growth phase-dependent translational control in the two species Halobacterium salinarum and Haloferax volcanii were as high as in eukaryotes. However, nothing is known about the mechanisms of translational regulation in archaea. Therefore, two genes exhibiting opposing directions of regulation were selected to unravel the importance of untranslated regions (UTRs) for differential translational control in vivo. Differential translational regulation in exponentially growing versus stationary phase cells was studied by comparing translational efficiencies using a reporter gene system. Translational regulation was not observed when 5'-UTRs or 3'-UTRs alone were fused to the reporter gene. However, their simultaneous presence was sufficient to transfer differential translational control from the native transcript to the reporter transcript. This was true for both directions of translational control. Translational regulation was completely abolished when stem loops in the 5'-UTR were changed by mutagenesis. An “UTR-swap” experiment demonstrated that the direction of translational regulation is encoded in the 3'-UTR, not in the 5'-UTR. While much is known about 5'-UTR-dependent translational control in bacteria, the reported findings provide the first examples that both 5'- and 3'-UTRs are essential and sufficient to drive differential translational regulation in a prokaryote and therefore have to functionally interact in vivo. The current results indicate that 3'-UTR-dependent translational control had already evolved before capping and polyadenylation of transcripts were invented, which are essential for circularization of transcripts in eukaryotes.
Background: Studies on the development of the nervous system and the musculature of invertebrates have become more sophisticated and numerous within the last decade and have proven to provide new insights into the evolutionary history of organisms. In order to provide new morphogenetic data on opisthobranch gastropods we investigated the neuromuscular development in the nudibranch Aeolidiella stephanieae Valdez, 2005 using immunocytochemistry as well as F-actin labelling in conjunction with confocal laser scanning microscopy (cLSM). Results: The ontogenetic development of Aeolidiella stephanieae can be subdivided into 8 stages, each recognisable by characteristic morphological and behavioural features as well as specific characters of the nervous system and the muscular system, respectively. The larval nervous system of A. stephanieae includes an apical organ, developing central ganglia, and peripheral neurons associated with the velum, foot and posterior, visceral part of the larva. The first serotonergic and FMRFamidergic neural structures appear in the apical organ that exhibits an array of three sensory, flask-shaped and two non-sensory, round neurons, which altogether disappear prior to metamorphosis. The postmetamorphic central nervous system (CNS) becomes concentrated, and the rhinophoral ganglia develop together with the anlage of the future rhinophores whereas oral tentacle ganglia are not found. The myogenesis in A. stephanieae begins with the larval retractor muscle followed by the accessory larval retractor muscle, the velar or prototroch muscles and the pedal retractors that all together degenerate during metamorphosis, and the adult muscle complex forms de novo. Conclusions: Aeolidiella stephanieae comprises features of the larval and postmetamorphic nervous as well as muscular system that represent the ground plan of the Mollusca or even the Trochozoa (e. g. presence of the prototrochal or velar muscle ring). On the one hand, A. stephanieae shows some features shared by all nudibranchs like the postmetamorphic condensation of the CNS, the possession of rhinophoral ganglia and the lack of oral tentacle ganglia as well as the de novo formation of the adult muscle complex. On the other hand, the structure and arrangement of the serotonergic apical organ is similar to other caenogastropod and opisthobranch gastropods supporting their sister group relationship.
A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage PhiC31. Host strains expressing the PhiC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetRgene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.
Myc-induced SUN domain–containing protein (Misu or NSun2) is a nucleolar RNA methyltransferase important for c-Myc–induced proliferation in skin, but the mechanisms by which Misu contributes to cell cycle progression are unknown. In this study, we demonstrate that Misu translocates from the nucleoli in interphase to the spindle in mitosis as an RNA–protein complex that includes 18S ribosomal RNA. Functionally, depletion of Misu caused multiple mitotic defects, including formation of unstructured spindles, multipolar spindles, and chromosome missegregation, leading to aneuploidy and cell death. The presence of both RNA and Misu is required for correct spindle assembly, and this process is independent of active translation. Misu might mediate its function at the spindle by recruiting nucleolar and spindle-associated protein (NuSAP), an essential microtubule-stabilizing and bundling protein. We further identify NuSAP as a novel direct target gene of c-Myc. Collectively, our results suggest a novel mechanism by which c-Myc promotes proliferation by stabilizing the mitotic spindle in fast-dividing cells via Misu and NuSAP.
The high sensitivity of male reproductive cells to high temperatures may be due to an inadequate heat stress response. The results of a comprehensive expression analysis of HsfA2 and Hsp17-CII, two important members of the heat stress system, in the developing anthers of a heat-tolerant tomato genotype are reported here. A transcriptional analysis at different developmental anther/pollen stages was performed using semi-quantitative and real-time PCR. The messengers were localized using in situ RNA hybridization, and protein accumulation was monitored using immunoblot analysis. Based on the analysis of the gene and protein expression profiles, HsfA2 and Hsp17-CII are finely regulated during anther development and are further induced under both short and prolonged heat stress conditions. These data suggest that HsfA2 may be directly involved in the activation of protection mechanisms in the tomato anther during heat stress and, thereby, may contribute to tomato fruit set under adverse temperatures. Key words: Anther development, heat stress, HsfA2, Hsp17-CII, pollen, tomato
Crista junctions (CJs) are important for mitochondrial organization and function, but the molecular basis of their formation and architecture is obscure. We have identified and characterized a mitochondrial membrane protein in yeast, Fcj1 (formation of CJ protein 1), which is specifically enriched in CJs. Cells lacking Fcj1 lack CJs, exhibit concentric stacks of inner membrane in the mitochondrial matrix, and show increased levels of F1FO–ATP synthase (F1FO) supercomplexes. Overexpression of Fcj1 leads to increased CJ formation, branching of cristae, enlargement of CJ diameter, and reduced levels of F1FO supercomplexes. Impairment of F1FO oligomer formation by deletion of its subunits e/g (Su e/g) causes CJ diameter enlargement and reduction of cristae tip numbers and promotes cristae branching. Fcj1 and Su e/g genetically interact. We propose a model in which the antagonism between Fcj1 and Su e/g locally modulates the F1FO oligomeric state, thereby controlling membrane curvature of cristae to generate CJs and cristae tips.
Diffusion of inner membrane proteins is a prerequisite for correct functionality of mitochondria. The complicated structure of tubular, vesicular or flat cristae and their small connections to the inner boundary membrane impose constraints on the mobility of proteins making their diffusion a very complicated process. Therefore we investigate the molecular transport along the main mitochondrial axis using highly accurate computational methods. Diffusion is modeled on a curvilinear surface reproducing the shape of mitochondrial inner membrane (IM). Monte Carlo simulations are carried out for topologies resembling both tubular and lamellar cristae, for a range of physiologically viable crista sizes and densities. Geometrical confinement induces up to several-fold reduction in apparent mobility. IM surface curvature per se generates transient anomalous diffusion (TAD), while finite and stable values of projected diffusion coefficients are recovered in a quasi-normal regime for short- and long-time limits. In both these cases, a simple area-scaling law is found sufficient to explain limiting diffusion coefficients for permeable cristae junctions, while asymmetric reduction of the junction permeability leads to strong but predictable variations in molecular motion rate. A geometry-based model is given as an illustration for the time-dependence of diffusivity when IM has tubular topology. Implications for experimental observations of diffusion along mitochondria using methods of optical microscopy are drawn out: a non-homogenous power law is proposed as a suitable approach to TAD. The data demonstrate that if not taken into account appropriately, geometrical effects lead to significant misinterpretation of molecular mobility measurements in cellular curvilinear membranes.
Oscillating magnetic field disrupts magnetic orientation in Zebra finches, Taeniopygia guttata
(2009)
Background Zebra finches can be trained to use the geomagnetic field as a directional cue for short distance orientation. The physical mechanisms underlying the primary processes of magnetoreception are, however, largely unknown. Two hypotheses of how birds perceive magnetic information are mainly discussed, one dealing with modulation of radical pair processes in retinal structures, the other assuming that iron deposits in the upper beak of the birds are involved. Oscillating magnetic fields in the MHz range disturb radical pair mechanisms but do not affect magnetic particles. Thus, application of such oscillating fields in behavioral experiments can be used as a diagnostic tool to decide between the two alternatives. Methods In a setup that eliminates all directional cues except the geomagnetic field zebra finches were trained to search for food in the magnetic north/south axis. The birds were then tested for orientation performance in two magnetic conditions. In condition 1 the horizontal component of the geomagnetic field was shifted by 90 degrees using a helmholtz coil. In condition 2 a high frequently oscillating field (1.156 MHz) was applied in addition to the shifted field. Another group of birds was trained to solve the orientation task, but with visual landmarks as directional cue. The birds were then tested for their orientation performance in the same magnetic conditions as applied for the first experiment. Results The zebra finches could be trained successfully to orient in the geomagnetic field for food search in the north/south axis. They were also well oriented in test condition 1, with the magnetic field shifted horizontally by 90 degrees. In contrast, when the oscillating field was added the directional choices during food search were randomly distributed. Birds that were trained to visually guided orientation showed no difference of orientation performance in the two magnetic conditions.