Protein–nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR

DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein–DNA and protein–ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein–DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy. Interactions are established by DNP-enhanced ³¹P–¹³C polarization-transfer experiments followed by the recording of a 2D ¹³C–¹³C correlation experiment. The NMR spectra were obtained in less than 2 days and allowed the identification of residues of the motor protein involved in nucleotide binding.     

Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster

Hairless (H) is the major antagonist within the Notch signalling pathway of Drosophila melanogaster. By binding to Suppressor of Hairless [Su(H)] and two co-repressors, H induces silencing of Notch target genes in the absence of Notch signals. We have applied genomic engineering to create several new H alleles. To this end the endogenous H locus was replaced with an attP site by homologous recombination, serving as a landing platform for subsequent site directed integration of different H constructs. This way we generated a complete H knock out allele H ^attP, reintroduced a wild type H genomic and a cDNA-construct (H ^gwt, H ^cwt) as well as two constructs encoding H proteins defective of Su(H) binding (H ^LD, H ^iD). Phenotypes regarding viability, bristle and wing development were recorded, and the expression of Notch target genes wingless and cut was analysed in mutant wing discs or in mutant cell clones. Moreover, genetic interactions with Notch (N ⁵⁴¹⁹) and Delta (Dl ^B2) mutants were addressed. Overall, phenotypes were largely as expected: both H ^LD and H ^iD were similar to the H ^attP null allele, indicating that most of H activity requires the binding of Su(H). Both rescue constructs H ^gwt and H ^cwt were homozygous viable without phenotype. Unexpectedly, the hemizygous condition uncovered that they were not identical to the wild type allele: notably H ^cwt showed a markedly reduced activity, suggesting the presence of as yet unidentified regulatory or stabilizing elements in untranslated regions of the H gene. Interestingly, H ^gwt homozygous cells expressed higher levels of H protein, perhaps unravelling gene-by-environment interactions.     

An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

The Fibrin Matrix Regulates Angiogenic Responses within the Hemostatic Microenvironment through Biochemical Control

Conceptually, premature initiation of post-wound angiogenesis could interfere with hemostasis, as it relies on fibrinolysis. The mechanisms facilitating orchestration of these events remain poorly understood, however, likely due to limitations in discerning the individual contribution of cells and extracellular matrix. Here, we designed an in vitro Hemostatic-Components-Model (HCM) to investigate the role of the fibrin matrix as protein factor-carrier, independent of its cell-scaffold function. After characterizing the proteomic profile of HCM-harvested matrix releasates, we demonstrate that the key pro-/anti-angiogenic factors, VEGF and PF4, are differentially bound by the matrix. Changing matrix fibrin mass consequently alters the balance of releasate factor concentrations, with differential effects on basic endothelial cell (EC) behaviors. While increasing mass, and releasate VEGF levels, promoted EC chemotactic migration, it progressively inhibited tube formation, a response that was dependent on PF4. These results indicate that the clot’s matrix component initially serves as biochemical anti-angiogenic barrier, suggesting that post-hemostatic angiogenesis follows fibrinolysis-mediated angiogenic disinhibition. Beyond their significance towards understanding the spatiotemporal regulation of wound healing, our findings could inform the study of other pathophysiological processes in which coagulation and angiogenesis are prominent features, such as cardiovascular and malignant disease.     

Immobilisation of phosphorus by iron-coated roots of submerged macrophytes

Hydrobiologia - To observe effects on the phosphorus retention mechanisms of a lake after re-colonisation by macrophytes, Potamogeton crispus L. and Elodea canadensis Michx. were planted in lab...     

Long-term effects of dietary isoflavones on uterine gene expression profiles

Isoflavones (ISOs) are bioactive food ingredients of the traditional East Asian diet and currently discussed as alternatives to classical hormone replacement therapies and for reducing the prevalence of hormone-dependent cancers. Although there are many studies on ISOs, not much is known about their long-term effects.Therefore, we performed an animal experiment analyzing the effects of three different diets: a phytoestrogen-free diet, a diet supplemented with genistein (700 μg/g diet) and an ISO-high diet (232 μg daidzein and 240 μg genistein/g) at two distinct time points, juvenile (21 days) and adult (97 days). Exposure started prior to mating of the parents and throughout the life of the offspring.We observed a stronger increase of uterine wet weights in juvenile offspring with genistein exposure (1018 ± 350 mg/kg BW) than with ISO-high diet (497 ± 133 mg/kg BW). Whereas the expression of proliferation related genes (PCNA; Ki67; IGF-1; IGF-1R), analyzed by real-time-qPCR and Western blot, were significantly down-regulated in juvenile animals exposed to genistein. Additionally, genistein exposure led to estrogenic responses, observed upon increase of complement C3 and decrease of estrogen receptors gene expressions, while the exposure to ISO-high diet did not show these effects.In conclusion, both the time point on which phytoestrogen exposure starts together with the composition of the ingested phytoestrogen containing diet are of great importance for the biological response of the offspring.     

Cloning and enzymatic characterization of four thermostable fungal endo-1,4-β-xylanases

Endo-1,4-β-xylanases (EC 3.2.1.8) hydrolyze the 1,4-β-D-xylosidic linkages in xylans, the most abundant hemicellulose in plant cell walls. Xylanase enzymes have numerous industrial applications, including the manufacturing of animal feed, bread, juice and wine, pulp and paper, and biofuels. In this study, two glycosyl hydrolase family 10 members designated GtXyn10A and GtXyn10B and two glycosyl hydrolase family 11 members, OpXyn11A and CcXyn11C, were functionally expressed and subjected to biochemical characterization. The K _M , V _max, and k _cat values of the four xylanases, determined using birchwood xylan, ranged from 0.27 to 1.1 mg/mL, 130 to 980 μmol/min/mg, and 109 to 344 s^?1, respectively, where OpXyn11A gave the highest and GtXyn10B the lowest values for all three parameters. Substrate specificity studies and analysis of the products released during the degradation of xylo-oligosaccharides and three types of xylan revealed significant differences in catalytic properties, particularly between OpXyn11A and the other xylanases and between the family 10 and the family 11 xylanases. Molecular modeling suggests that the unique substrate specificity of OpXyn11A can be attributed to the presence of a serine rather that an asparagine or aspartate residue at the +1 substrate binding site. Additionally, all four xylanases exhibited biochemical characteristics of interest for various commercial applications.     

Long-Term GABA Administration Induces Alpha Cell-Mediated Beta-like Cell Neogenesis

1. Download high-res image (401KB)
2. Download full-size image

     

Process development of itaconic acid production by a natural wild type strain of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> to reach industrially relevant final titers

Itaconic acid is a promising organic acid and is commercially produced by submerged fermentation of Aspergillus terreus. The cultivation process of the sensitive filamentous fungus has been studied intensively since 1932, with respect to fermentation media components, oxygen supply, shearing rate, pH value, or culture method. Whereas increased final titers were achieved over the years, the productivity has so far remained quite low. In this study, the impact of the pH on the itaconic acid production was investigated in detail. The pH during the growth and production phase had a significant influence on the final itaconic acid concentration and pellet diameter. The highest itaconic acid concentration of 160 g/L was achieved at a 1.5-L scale within 6.7 days by raising and controlling the pH value to pH 3.4 in the production phase. An ammonia solution and an increased phosphate concentration were used with an itaconic acid yield of 0.46 (w/w) and an overall productivity of 0.99 g/L/h in a fed-batch mode. A cultivation with a lower phosphate concentration resulted in an equal final concentration with an increased yield of 0.58 (w/w) after 11.8 days and an overall productivity of 0.57 g/L/h. This optimized process was successfully transferred from a 1.5-L scale to a 15-L scale. After 9.7 days, comparable pellet morphology and a final concentration of 150 g/L itaconic acid was reached. This paper provides a process strategy to yield a final titer of itaconic acid from a wild-type strain of A. terreus which is in the same range as the well-known citric acid production.