Multi-targeted antifolates aimed at avoiding drug resistance form covalent closed inhibitory complexes with human and Escherichia coli thymidylate synthases.

Crystal structures of four pyrrolo(2,3-d)pyrimidine-based antifolate compounds, developed as inhibitors of thymidylate synthase (TS) in a strategy to circumvent drug-resistance, have been determined in complexes with their in vivo target, human thymidylate synthase, and with the structurally best-characterized Escherichia coli enzyme, to resolutions of 2.2-3.0 A. The 2.9 A crystal structure of a complex of human TS with one of the inhibitors, the multi-targeted antifolate LY231514, demonstrates that this compound induces a "closed" enzyme conformation and leads to formation of a covalent bond between enzyme and substrate. This structure is one of the first liganded human TS structures, and its solution was aided by mutation to facilitate crystallization. Structures of three other pyrrolo(2,3-d)pyrimidine-based antifolates in complex with Escherichia coli TS confirm the orientation of this class of inhibitors in the active site. Specific interactions between the polyglutamyl moiety and a positively charged groove on the enzyme surface explain the marked increase in affinity of the pyrrolo(2,3-d)pyrimidine inhibitors once they are polyglutamylated, as mediated in vivo by the cellular enzyme folyl polyglutamate synthetase.     

Chitin in the Silk Gland Ducts of the Spider Nephila edulis and the Silkworm Bombyx mori

Here we report the detection and localisation of chitin in the cuticle of the spinning ducts of both the spider Nephila edulis and the silkworm Bombyx mori. Our observations demonstrate that the duct walls of both animals contain chitin notwithstanding totally independent evolutionary pathways of the systems. We conclude that chitin may well be an essential component for the construction of spinning ducts; we further conclude that in both species chitin may indicate the evolutionary origin of the spinning ducts.     

Diversity of North American map and sawback turtles (Testudines: Emydidae: Graptemys)

Map turtles of the genus Graptemys are native to North America, where a high degree of drainage endemism is believed to have shaped current diversity. With 14 species and one additional subspecies, Graptemys represents the most diverse genus in the family Emydidae. While some Graptemys species are characterized by pronounced morphological differences, previous phylogenetic analyses have failed yet to confirm significant levels of genetic divergence for many taxa. As a consequence, it has been debated whether Graptemys is taxonomically inflated or whether the low genetic divergence observed reflects recent radiations or ancient hybridization. In this study, we analysed three mtDNA blocks (3228 bp) as well as 12 nuclear loci (7844 bp) of 89 specimens covering all species and subspecies of Graptemys. Our analyses of the concatenated mtDNA sequences reveal that the widespread G. geographica constitutes the sister taxon of all other Graptemys species. These correspond to two clades, one comprised of all broad‐headed Graptemys species and another clade containing the narrow‐headed species. Most species of the broad‐headed clade are reciprocally monophyletic, except for G. gibbonsi and G. pearlensis, which are not differentiated. By contrast, in the narrow‐headed clade, many currently recognized species are not monophyletic and divergence is significantly less pronounced. Haplotype networks of phased nuclear loci show low genetic divergence among taxa and many shared haplotypes. Principal component analyses using coded phased nuclear DNA sequences revealed eight distinct clusters within Graptemys that partially conflict with the terminal mtDNA clades. This might be explained by male‐mediated gene flow across drainage basins and female philopatry within drainage basins. Our results support that Graptemys is taxonomically oversplit and needs to be revised.     

Analysis of the protein kinase activity of moss phytochrome expressed in fibroblast cell culture

In the moss Ceratodon purpureus a phytochrome gene encodes a phytochrome type (PhyCer) which has a C-terminal domain homologous to the catalytic domain of eukaryotic protein kinases (PKs). PhyCer exhibits sequence conservation to serine/ threonine as well to tyrosine kinases. Since PhyCer is expressed very weakly in moss cells, to investigate the proposed PK activity of PhyCer, we overexpressed PhyCer transiently in fibroblast cells. For this purpose we made a chimeric receptor, EC-R, which consists of the extracellular, the membrane-spanning and the juxtamembrane domains of the human epidermal growth-factor receptor (EGF-R) linked to the PK catalytic domain of PhyCer (CerKin). The expression of EC-R in transiently transfected cells was confirmed with antibodies directed against the extracellular domain of EGF-R or against CerKin. Both EGF-R and EC-R were immunoprecipitated from lysates of overexpressing cells with antibodies against the extracellular domain of EGF-R. Phosphorylation experiments were performed with the immunoprecipitates and the phosphorylation products were subjected to phosphoamino acid analysis. Phosphorylation products specifically obtained with EC-R-transfected cells exhibit phosphorylation on serine and threonine residues. In EC-R transfected cells the endogenous EGF-R showed enhanced phosphorylation of serine and threonine residues compared to EGF-R immuno-precipitated from control cells. Although CerKin is closest to the catalytic domain of a protein tyrosine kinase from Dictyostelium discoideum, EC-R does not appear to phosphorylate tyrosine residues in vitro. From our data we conclude that PhyCer carries an active PK domain capable of phosphorylating serine and threonine residues.Abbreviations CerKin protein kinase catalytic domain of PhyCer - EC-R chimeric receptor consisting of the extracellular, the membrane spanning and the juxtamembrane domains of the human epidermal growth factor receptor (EGF-R) linked to the protein kinase catalytic domain of PhyCer - EGF-R epidermal growth factor receptor - mAb monoclonal antibody - PhyCer phytochrome gene in Ceratodon encoding a phytochrome type which has a C-terminal domain homologous to the catalytic domain of eucaryotic protein kinases - PK protein kinase - PVDF polyvinyl difluoride - Ser serine - Thr threonine - Tyr tyrosine Dr. Patricia Algarra was supported by the Alexander von Humboldt Foundation, Germany. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany.     

Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility

To develop adsorbents for the specific removal of tumor necrosis factor-alpha (TNF) in extracorporeal blood purification, cellulose microparticles were functionalized either with a monoclonal anti-TNF antibody (mAb) or with recombinant human antibody fragments (Fab). The TNF binding capacity of the adsorbents was determined with in vitro batch experiments using spiked human plasma (spike: 1200 pg TNF/mL; 1 mg particles in 250 muL plasma). Random immobilization of the full-sized monoclonal antibody to periodate-activated cellulose yielded particles with excellent adsorption capacity (258.1 +/- 48.6 pg TNF per mg adsorbent wet weight). No leaching of antibody was detectable, and the adsorbents retained their activity for at least 12 months at 4 degrees C. We found that the conditions used during immobilization of the antibody (pH, nature of the reducing agent) profoundly influenced the biocompatibility of the resulting adsorbents, especially with respect to activation of the complement system. Particles obtained by random immobilization of the monovalent Fab fragments on periodate-activated cellulose using the same conditions as for immobilization of the mAb exhibited only low adsorption capacity (44 +/- 7 pg/mg adsorbent wet weight). Oriented coupling of the Fab fragments on chelate-epoxy cellulose via a C-terminal histidine tag, however, increased the adsorption capacity to 178.3 +/- 8.6 pg TNF/mg adsorbent wet weight. Thus, in the case of small, monovalent ligands, the orientation on the carrier is critical to retain full binding activity.     

Automated UMLS-Based Comparison of Medical Forms

Medical forms are very heterogeneous: on a European scale there are thousands of data items in several hundred different systems. To enable data exchange for clinical care and research purposes there is a need to develop interoperable documentation systems with harmonized forms for data capture. A prerequisite in this harmonization process is comparison of forms. So far – to our knowledge – an automated method for comparison of medical forms is not available. A form contains a list of data items with corresponding medical concepts. An automatic comparison needs data types, item names and especially item with these unique concept codes from medical terminologies. The scope of the proposed method is a comparison of these items by comparing their concept codes (coded in UMLS). Each data item is represented by item name, concept code and value domain. Two items are called identical, if item name, concept code and value domain are the same. Two items are called matching, if only concept code and value domain are the same. Two items are called similar, if their concept codes are the same, but the value domains are different. Based on these definitions an open-source implementation for automated comparison of medical forms in ODM format with UMLS-based semantic annotations was developed. It is available as package compareODM from http://cran.r-project.org. To evaluate this method, it was applied to a set of 7 real medical forms with 285 data items from a large public ODM repository with forms for different medical purposes (research, quality management, routine care). Comparison results were visualized with grid images and dendrograms. Automated comparison of semantically annotated medical forms is feasible. Dendrograms allow a view on clustered similar forms. The approach is scalable for a large set of real medical forms.     

An SAR sequence containing 395 bp DNA fragment mediates enhanced,gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants

A 395 bp fragment located downstream from the soybean heat shock geneGmhsp 17.6-L exhibits several characteristics of scaffold attachment region (SAR) sequences. It contains matrix consensus elements, a topoisomerase II binding sequence and it associates with the isolated nuclear scaffold of soybeanin vitro. Chimaeric genes containing the SAR_L fragment either at one side (5 or 3) or at both sides of a heat shock promoter-regulated -glucuronidase reporter gene were constructed. A five-to nine-fold increase of heat-inducible -glucuronidase activity was observed in transgenic tobacco plants containing constructs with SAR_L fragments either at both sides or with at least one SAR_L copy located upstream from the reporter gene. The gene copy number is positively correlated with the level of heat-inducible reporter gene activity in these. plants but positional effects are not entirely eliminated. Thus, SAR sequences may potentially be used to increase gene expression, via as yet unknown mechanisms, and to reduce adverse effects on the expression of multiple gene copies in transgenic plants.