A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes

The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection significantly improves the analytical results in terms of higher number of detected phosphorylation sites and spectra of higher quality. Metal oxide chromatography (MOC) has been recently described for selective phosphopeptide enrichment (Pinkse et al., 2004 [1]; Larsen et al., 2005 [2]; Kweon and Hakansson, 2006 [3]; Cantin et al., 2007 [4]; Collins et al., 2007 [5]). In the present work we have tested the effect of a modified loading solvent containing a novel acid mix and optimized wash conditions on the efficiency of TiO₂-based phosphopeptide enrichment in order to improve our previously published method (Mazanek et al., 2007 [6]). Applied to a test mixture of synthetic and BSA-derived peptides, the new method showed improved selectivity for phosphopeptides, whilst retaining a high recovery rate. Application of the new enrichment method to digested purified protein complexes resulted in the identification of a significantly higher number of phosphopeptides as compared to the previous method. Additionally, we have compared the performance of TiO₂ and ZrO₂ columns for the isolation and identification of phosphopeptides from purified protein complexes and found that for our test set, both media performed comparably well. In summary, our improved method is highly effective for the enrichment of phosphopeptides from purified protein complexes prior to mass spectrometry, and is suitable for large-scale phosphoproteomic projects that aim to elucidate phosphorylation-dependent cellular processes.     

EFFECTS OF INCREASED TEMPERATURE AND CO2 ON PHOTOSYNTHESIS,GROWTH, AND ELEMENTAL RATIOS IN MARINE SYNECHOCOCCUS AND PROCHLOROCOCCUS (CYANOBACTERIA)1

Little is known about the combined impacts of future CO₂ and temperature increases on the growth and physiology of marine picocyanobacteria. We incubated Synechococcus and Prochlorococcus under present‐day (380 ppm) or predicted year‐2100 CO₂ levels (750 ppm), and under normal versus elevated temperatures (+4°C) in semicontinuous cultures. Increased temperature stimulated the cell division rates of Synechococcus but not Prochlorococcus. Doubled CO₂ combined with elevated temperature increased maximum chl a–normalized photosynthetic rates of Synechococcus four times relative to controls. Temperature also altered other photosynthetic parameters (α, Φ_max, E_k, and ) in Synechococcus, but these changes were not observed for Prochlorococcus. Both increased CO₂ and temperature raised the phycobilin and chl a content of Synechococcus, while only elevated temperature increased divinyl chl a in Prochlorococcus. Cellular carbon (C) and nitrogen (N) quotas, but not phosphorus (P) quotas, increased with elevated CO₂ in Synechococcus, leading to ～20% higher C:P and N:P ratios. In contrast, Prochlorococcus elemental composition remained unaffected by CO₂, but cell volume and elemental quotas doubled with increasing temperature while maintaining constant stoichiometry. Synechococcus showed a much greater response to CO₂ and temperature increases for most parameters measured, compared with Prochlorococcus. Our results suggest that global change could influence the dominance of Synechococcus and Prochlorococcus ecotypes, with likely effects on oligotrophic food‐web structure. However, individual picocyanobacteria strains may respond quite differently to future CO₂ and temperature increases, and caution is needed when generalizing their responses to global change in the ocean.     

Adenoviral transduction is more efficient in alginate-derived chondrocytes than in monolayer chondrocytes

Gene transfer into cultured chondrocytes by using adenoviral vectors has potential applications in treating cartilage disorders. The present study was undertaken to compare and optimize two chondrocyte culture conditions for adenoviral transduction efficacy by using primary human articular chondrocytes cultivated either directly in a monolayer condition or as outgrowths from alginate-stored chondrocyte cultures. Isolated primary chondrocytes from human articular cartilage were either immediately transduced with an EGFP (enhanced green fluorescent protein)-gene-bearing adenoviral vector (1,000 and 3,000 virus particles/cell) or cultured in alginate before transduction. Immunohistochemistry and flow cytometric analysis were employed to determine the expression of extracellular matrix proteins and of the αvβ5 integrin receptor involved in adenoviral cell entry. Monolayer chondrocytes exhibited moderate transduction rates (mean 22.2% and 46.9% EGFP-positive cells at 1,000 and 3,000 virus particles/cell by 72 h post-transduction), whereas alginate-derived chondrocytes revealed significantly higher transduction efficacies (95.7% and 99%). Both monolayer and alginate-derived chondrocytes expressed αvβ5 integrin, type II collagen and cartilage proteoglycans. The mean fluorescence intensity of type II collagen was significantly higher in the alginate-derived chondrocytes, whereas that of αvβ5 integrin was higher in the monolayer chondrocytes. Our results indicate that transduction efficacy is independent of αvβ5 integrin expression levels in chondrocytes. Moreover, adenoviral transduction of alginate-derived chondrocytes is more efficient than that for monolayer chondrocytes and may be a suitable tool to achieve sufficient numbers of transduced and differentiated chondrocytes for experimental applications and cartilage repair. Dr. Gundula Schulze-Tanzil is supported by a grant awarded by the Rahel Hirsh Foundation from the Charité Medical Schools Berlin. The study was supported by a grant from the Deutsche Arthrosehilfe e.V.     

Systematic variation in mRNA 3'-processing signals during mouse spermatogenesis

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3′-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3′-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3′-processing characteristics in the testicular samples, compared to control sets of widely used 3′-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3′-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3′-untranslated regions (3′-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3′-UTR truncation and no significant difference in 3′-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3′-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis.     

Synthesis and biodistribution of new radiolabeled high-affinity choline transporter inhibitors [11C]hemicholinium-3 and [18F]hemicholinium-3

The high-affinity choline transporter (CHT1) system is an attractive target for the development of positron emission tomography (PET) biomarkers to probe brain, cardiac, and cancer diseases. An efficient and convenient synthesis of new radiolabeled CHT1 inhibitors [(11)C]hemicholinium-3 and [(18)F]hemicholinium-3 by solid-phase extraction (SPE) technique using a cation-exchange CM Sep-Pak cartridge has been well developed. The preliminary evaluation of both tracers through biodistribution studies in 9L-glioma rats has been performed, and the uptakes in the heart and tumor were observed, while very low brain uptake was seen.     

Dual pro-drugs of 2'-C-methyl guanosine monophosphate as potent and selective inhibitors of hepatitis C virus

We have previously reported the power of combining a 5'-phosphoramidate ProTide, phosphate pro-drug, motif with a 6-methoxy purine pro-drug entity to generate highly potent anti-HCV agents, leading to agents in clinical trial. We herein extend this work with the disclosure that a variety of alternative 6-substituents are tolerated. Several compounds exceed the potency of the prior 6-methoxy leads, and in almost every case the ProTide is several orders of magnitude more potent than the parent nucleoside. We also demonstrate that these agents act as pro-drugs of 2'-C-methyl guanosine monophosphate. We have also reported the novel use of hepatocyte cell lysate as an ex vivo model for ProTide metabolism.     

Design, synthesis, and activity of achiral analogs of 2-quinolones and indoles as non-thiol farnesyltransferase inhibitors

Beginning with the structure of tipifarnib (1), a series of inhibitors of FTase have been synthesized by transposition of the D-ring to the imidazole and subsequent modification of the 2-quinolone motif. The compounds in the new series may be achiral and have structural features that allow for analogs that are difficult or impossible to make in the tertiary carbon-based tipifarnib series. The most potent compound (4d) is 4 times more active in vitro against FTase than tipifarnib.