Organizing protein-DNA hybrids as nanostructures with programmed functionalities

The structural and functional information encoded in the base sequence of nucleic acids provides a means to organize hybrid protein-DNA nanostructures with pre-designed, programmed functionality. This review discusses the activation of enzyme cascades in supramolecular DNA-protein hybrid structures, the bioelectrocatalytic activation of redox enzymes on DNA scaffolds, and the programmed positioning of enzymes on 1D, 2D and 3D DNA nanostructures. These systems provide starting points towards the design of interconnected enzyme networks. Substantial progress in the tailoring of functional protein-DNA nanostructures has been accomplished in recent years, and advances in this field warrant a comprehensive discussion. The application of these systems for the control of biocatalytic transformations, for amplified biosensing, and for the synthesis of metallic nanostructures are addressed, and future prospects for these systems are highlighted.     

25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand

25-Hydroxyvitamin D₃ 1α-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D₃ into 1α,25-dihydroxyvitamin D₃, a vitamin D receptor ligand. 25-Hydroxyvitamin D₃ has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1α-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D₃ has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D₃ internalisation, in Cyp27b1^?/? cells explains their higher sensitivity to 25-hydroxyvitamin D₃. 25-Hydroxyvitamin D₃ action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D₃. Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D₃ in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D₃ with 1α,25-dihydroxyvitamin D₃ in Cyp27b1^?/? cells further demonstrates the agonistic action of 25-hydroxyvitamin D₃ and suggests that a synergism between 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ might be physiologically important. In conclusion, 25-hydroxyvitamin D₃ is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.     

Novel tetrahydrochinoline derived CETP inhibitors

In the course of our efforts to identify orally active cholesteryl ester transfer protein (CETP) inhibitors, we have continued to explore tetrahydrochinoline derivatives. Based on BAY 19-4789 structural modifications led to the discovery of novel cycloalkyl substituted compounds. Thus, example 11b is a highly potent CETP inhibitor both in vitro and in vivo in transgenic mice with favourable pharmacokinetic properties for clinical development.     

Phosphoinositide 3-kinase γ has Multiple Phospholipid Binding Sites

Phosphoinositide 3-kinase γ is a multifunctional enzyme with lipid and protein kinase activities that also acts as a scaffold protein in many diverse signalling processes. The enzyme contains five different domains, but their individual contributions to membrane binding are not fully understood. Here, using in vitro liposome binding assays of individual domains and deletion constructs of human phosphoinositide 3-kinase γ, we show that each domain is capable of binding anionic phospholipids to varying degrees, depending on the charge of the anionic substrate. Moreover, with the exception of the C2-domain, deletion of any single protein domain results in a complete loss of kinase activity toward both lipids and proteins.     

TaqMan Real-Time PCR Assays To Assess Arbuscular Mycorrhizal Responses to Field Manipulation of Grassland Biodiversity: Effects of Soil Characteristics,Plant Species Richness,and Functional Traits

Large-scale (temporal and/or spatial) molecular investigations of the diversity and distribution of arbuscular mycorrhizal fungi (AMF) require considerable sampling efforts and high-throughput analysis. To facilitate such efforts, we have developed a TaqMan real-time PCR assay to detect and identify AMF in environmental samples. First, we screened the diversity in clone libraries, generated by nested PCR, of the nuclear ribosomal DNA internal transcribed spacer (ITS) of AMF in environmental samples. We then generated probes and forward primers based on the detected sequences, enabling AMF sequence type-specific detection in TaqMan multiplex real-time PCR assays. In comparisons to conventional clone library screening and Sanger sequencing, the TaqMan assay approach provided similar accuracy but higher sensitivity with cost and time savings. The TaqMan assays were applied to analyze the AMF community composition within plots of a large-scale plant biodiversity manipulation experiment, the Jena Experiment, primarily designed to investigate the interactive effects of plant biodiversity on element cycling and trophic interactions. The results show that environmental variables hierarchically shape AMF communities and that the sequence type spectrum is strongly affected by previous land use and disturbance, which appears to favor disturbance-tolerant members of the genus Glomus. The AMF species richness of disturbance-associated communities can be largely explained by richness of plant species and plant functional groups, while plant productivity and soil parameters appear to have only weak effects on the AMF community.Arbuscular mycorrhizae are mutualistic associations between roots of plants and fungi that have been present for more than 400 million years (54). Approximately 80% of examined land plants (71), and almost all fungi of the phylum Glomeromycota (60), are capable of forming such associations. The main benefit of this relationship for plants is that it facilitates their acquisition of nutrients (especially P and N), while the fungus receives photoassimilates (7, 62). About 200 Glomeromycota species have been described to date, based on spore morphology (http://www.lrz-muenchen.de/∼schuessler/amphylo/amphylogeny.html), but there is increasing molecular evidence of significantly higher diversity in arbuscular mycorrhizal fungi (AMF) (10, 72).Diverse AMF communities have been detected in a wide range of plant communities (inter alia grasslands, boreal forests, and tropical communities; for an overview, see reference 48). Hence, AMF have been considered to be tolerant of wide ranges of ecological conditions and capable of associating with diverse plant partners. Identifying the factors regulating their community assemblages is challenging, but AMF community composition has been shown to be influenced by plant species diversity (e.g., see references 10, 22, and 33), and conversely, significant effects of AMF species and communities on the diversity and productivity of plant communities have been described (25, 68). Soil physicochemical parameters like phosphorus, nitrogen, and carbon availability (e.g., see references 4, 9, and 31); pH (17); moisture content (53); and disturbance (30) also reportedly influence AMF distribution. Hence, there is some support for niche theory, which presumes that two species of the same trophic level cannot coexist in a limited system and, if two species are present in such circumstances, one should become extinct (21). As a corollary, two cooccurring species must occupy niches that differ in some dimensions, e.g., plant hosts and/or soil properties (28). However, there are also indications that neutral ecological processes, as well as niche-defining parameters, may influence AMF diversity and community composition (17, 39). In contrast to niche theory, neutral theory (27) postulates that all individuals of every species at a given trophic level in a food web have ecological equivalence, and thus, all species within trophically defined communities can be regarded as open nonequilibrium assemblages that are solely shaped by dispersal and distinctions in spatiotemporal dimensions. According to the work of Hubbell (27), neutrality is defined at the level of individual organisms with identical probabilities of birth, death, migration, and speciation and not at the species level. In order to explore AMF communities more thoroughly and to test competing hypotheses, such as those raised by the niche and neutral theories, robust methods for high-throughput analyses of the communities are required.Recent investigations of variables that affect the structure of AMF communities have considered relationships between niche-defining dimensions, such as soil types (39) and pH gradients (17), and spatial variations in AMF community structure but not the role of plant diversity or functional traits of host plants. There have been several plant diversity manipulation experiments designed for coanalyzing multiple sets of ecological variables (e.g., the BIODEPTH and Cedar Creek projects) that would have been ideal for detailed examinations of effects of ecological variables on AMF, but previously reported AMF analyses in these experiments have been limited to counts of spores in a single study (11). However, not all AMF species regularly sporulate, and when present, spores poorly reflect AMF diversity (69), since active AMF occur as mycelia in roots and soils (e.g., see references 12 and 26). PCR-based molecular techniques enable much more rigorous characterization of AMF communities in these compartments (e.g., see references 26, 36, and 72), but assessments of broad spatial (42) and/or temporal (52) variations in AMF communities require analysis of large numbers of samples, which is not feasible using conventional PCR amplification followed by cloning and sequencing. This challenge can be potentially met by real-time PCR-based approaches, in which the AMF sequence types present in compartments of interest are first identified and then sequence type-specific probes are used for large-scale screening in real-time PCR TaqMan assays.In the study presented here, we explored AMF diversity in plots used in the Jena Experiment, a grassland plant diversity manipulation of 60 plant species representing four functional groups in 81 plots of 400 m² (56). The overall AMF diversity and community structure were first assessed by PCR amplification, cloning, and sequencing (55) of internal transcribed spacer (ITS) ribosomal DNA (rDNA) gene sequences in soil samples from 23 representative plots. Using the acquired data, we then developed sequence type-specific probes, which were applied in high-throughput real-time PCR TaqMan assays of samples from all 81 experimental plots, and the effects of 15 plant and soil variables on the AMF community assemblage were investigated.     

Genomic microstructure and differential expression of the genes encoding UDP-glucose:sinapate glucosyltransferase (UGT84A9) in oilseed rape (Brassica napus)

In oilseed rape (Brassica napus), the glucosyltransferase UGT84A9 catalyzes the formation of 1-O-sinapoyl-β-glucose, which feeds as acyl donor into a broad range of accumulating sinapate esters, including the major antinutritive seed component sinapoylcholine (sinapine). Since down-regulation of UGT84A9 was highly efficient in decreasing the sinapate ester content, the genes encoding this enzyme were considered as potential targets for molecular breeding of low sinapine oilseed rape. B. napus harbors two distinguishable sequence types of the UGT84A9 gene designated as UGT84A9-1 and UGT84A9-2. UGT84A9-1 is the predominantly expressed variant, which is significantly up-regulated during the seed filling phase, when sinapate ester biosynthesis exhibits strongest activity. In the allotetraploid genome of B. napus, UGT84A9-1 is represented by two loci, one derived from the Brassica C-genome (UGT84A9a) and one from the Brassica A-genome (UGT84A9b). Likewise, for UGT84A9-2 two loci were identified in B. napus originating from both diploid ancestor genomes (UGT84A9c, Brassica C-genome; UGT84A9d, Brassica A-genome). The distinct UGT84A9 loci were genetically mapped to linkage groups N15 (UGT84A9a), N05 (UGT84A9b), N11 (UGT84A9c) and N01 (UGT84A9d). All four UGT84A9 genomic loci from B. napus display a remarkably low micro-collinearity with the homologous genomic region of Arabidopsis thaliana chromosome III, but exhibit a high density of transposon-derived sequence elements. Expression patterns indicate that the orthologous genes UGT84A9a and UGT84A9b should be considered for mutagenesis inactivation to introduce the low sinapine trait into oilseed rape.     

Spatial temperature distribution in human hairy and glabrous skin after infrared CO<Subscript>2</Subscript> laser radiation

Background  

CO₂ lasers have been used for several decades as an experimental non-touching pain stimulator. The laser energy is absorbed by the water content in the most superficial layers of the skin. The deeper located nociceptors are activated by passive conduction of heat from superficial to deeper skin layers.