High-Level Expression of Various Apolipoprotein (a) Isoforms by "Transferrinfection". The Role of Kringle IV Sequences in the Extracellular Association with Low-Density Lipoprotein

Characterization of the assembly of lipoprotein(a) [Lp(a)] is of fundamental importance to understanding the biosynthesis and metabolism of this atherogenic lipoprotein. Since no established cell lines exist that express Lp(a) or apolipoprotein(a) [apo(a)], a "transferrinfection" system for apo(a) was developed utilizing adenovirus receptor- and transferrin receptor-mediated DNA uptake into cells. Using this method, different apo(a) cDNA constructions of variable length, due to the presence of 3, 5, 7, 9, 15, or 18 internal kringle IV sequences, were expressed in cos-7 cells or CHO cells. All constructions contained kringle IV-36, which includes the only unpaired cysteine residue (Cys-4057) in apo(a). r-Apo(a) was synthesized as a precursor and secreted as mature apolipoprotein into the medium. When medium containing r-apo(a) with 9, 15, or 18 kringle IV repeats was mixed with normal human plasma LDL, stable complexes formed that had a bouyant density typical of Lp(a). Association was substantially decreased if Cys-4057 on r-apo(a) was replaced by Arg by site-directed mutagenesis or if Cys-4057 was chemically modified. Lack of association was also observed with r-apo(a) containing only 3, 5, or 7 kringle IV repeats without "unique kringle IV sequences", although Cys-4057 was present in all of these constructions. Synthesis and secretion of r-apo(a) was not dependent on its sialic acid content. r-Apo(a) was expressed even more efficiently in sialylation-defective CHO cells than in wild-type CHO cells. In transfected CHO cells defective in the addition of N-acetylglucosamine, apo(a) secretion was found to be decreased by 50%. Extracellular association with LDL was not affected by the carbohydrate moiety of r-apo(a), indicating a protein-protein interaction between r-apo(a) and apoB. These results show that, besides kringle IV-36, other kringle IV sequences are necessary for the extracellular association of r-apo(a) with LDL. Changes in the carbohydrate moiety of apo(a), however, do not affect complex formation

Stability of Terrestrial Planets in the Habitable Zone of Gl 777 A, HD 72659, Gl 614, 47 Uma and HD 4208

We have undertaken a thorough dynamical investigation of five extrasolar planetary systems using extensive numerical experiments. The systems Gl 777 A, HD 72659, Gl 614, 47 Uma and HD 4208 were examined concerning the question of whether they could host terrestrial like planets in their habitable zones (=HZ). First we investigated the mean motion resonances between fictitious terrestrial planets and the existing gas giants in these five extrasolar systems. Then a fine grid of initial conditions for a potential terrestrial planet within the HZ was chosen for each system, from which the stability of orbits was then assessed by direct integrations over a time interval of 1 million years. The computations were carried out using a Lie-series integration method with an adaptive step size control. This integration method achieves machine precision accuracy in a highly efficient and robust way, requiring no special adjustments when the orbits have large eccentricities. The stability of orbits was examined with a determination of the Renyi entropy, estimated from recurrence plots, and with a more straight forward method based on the maximum eccentricity achieved by the planet over the 1 million year integration. Additionally, the eccentricity is an indication of the habitability of a terrestrial planet in the HZ; any value of e>0.2 produces a significant temperature difference on a planet's surface between apoapse and periapse. The results for possible stable orbits for terrestrial planets in habitable zones for the five systems are summarized as follows: for Gl 777 A nearly the entire HZ is stable, for 47 Uma, HD 72659 and HD 4208 terrestrial planets can survive for a sufficiently long time, while for Gl 614 our results exclude terrestrial planets moving in stable orbits within the HZ.Comment: 14 pages, 18 figures submitted to A&

Negotiating care in the context of Finnish and Italian elder care policies

Negotiation is an integral part of all elder care, which by definition involves a relation between at least two people. In this article we analyse negotiations concerning elder care in the context of Finnish and Italian elder care policies. At the macro level negotiations on elder care are shaped by elder care policies and at the micro level by individual skills and resources. Our focus is on the negotiations on eligibility that take place when elders attempt to access care. The data consist of qualitative interviews with Finnish and Italian elders in need of care. The analysis of individual experiences of care negotiations reflects the implementation of elder care policies. The results indicate that the most negotiated eligibility criteria when seeking access to elder care are need, money and social relations. These criteria are negotiated when seeking eligibility to different sources of care: informal care, grey market, market-based, non-profit and public services. In Italy, negotiation is particularly crucial when accessing grey market care. Cash as the main Italian elder care policy tool tends to enhance the role of and need for negotiation. In Finland, a greater part of elder care is provided by the public sector and therefore the process of negotiation is more standardized than in Italy

Pigment Epithelium–Derived Factor Regulates Lipid Metabolism via Adipose Triglyceride Lipase

OBJECTIVE: Pigment epithelium-derived factor (PEDF) is an adipocyte-secreted factor involved in the development of insulin resistance in obesity. Previous studies have identified PEDF as a regulator of triacylglycerol metabolism in the liver that may act through adipose triglyceride lipase (ATGL). We used ATGL(-/-) mice to determine the role of PEDF in regulating lipid and glucose metabolism. RESEARCH DESIGN AND METHODS: Recombinant PEDF was administered to ATGL(-/-) and wild-type mice, and whole-body energy metabolism was studied by indirect calorimetry. Adipose tissue lipolysis and skeletal muscle fatty acid metabolism was determined in isolated tissue preparations. Muscle lipids were assessed by electrospray ionization-tandem mass spectrometry. Whole-body insulin sensitivity and skeletal muscle glucose uptake were assessed. RESULTS: PEDF impaired the capacity to adjust substrate selection, resulting in a delayed diurnal decline in the respiratory exchange ratio, and suppressed daily fatty acid oxidation. PEDF enhanced adipocyte lipolysis and triacylglycerol lipase activity in skeletal muscle. Muscle fatty acid uptake and storage were unaffected, whereas fatty acid oxidation was impaired. These changes in lipid metabolism were abrogated in ATGL(-/-) mice and were not attributable to hypothalamic actions. ATGL(-/-) mice were also refractory to PEDF-mediated insulin resistance, but this was not related to changes in lipid species in skeletal muscle. CONCLUSIONS: The results are the first direct demonstration that 1) PEDF influences systemic fatty acid metabolism by promoting lipolysis in an ATGL-dependent manner and reducing fatty acid oxidation and 2) ATGL is required for the negative effects of PEDF on insulin action

Österreichisches Projekt Grundlagen zur Züchtung, Vermehrung und Sorten-/Saatgutprüfung für den Biolandbau: Ergebnisübersicht

In the Austrian research project “Basic principles for breeding, multiplication and variety testing for organic agriculture” (from 2004 until 2008) novel methods for the assessment of the suitability of seed and cultivars for organic farming were developed and evaluated by an interdisciplinary co-operation of researchers, breeders and variety testers. Organic farming requires specific combinations of crop plant characters, especially seed health and resistance against seed-borne diseases. Another crucial feature is the competitive ability against weeds. Early development was found to significantly increase the competitiveness of different crop plants. Genotypic variation in characters necessary for an efficient utilisation of below-ground resources was also investigated, e.g. interrelations between root development and drought stress tolerance and between mycorrhiza formation and nutrient use efficiency. Based on the results obtained in various cultivar trials, it can be concluded that genotypes suitable for organic growing conditions may be pre-selected from early breeding material of conventional breeding programmes. The selected breeding material must subsequently be rigorously tested on organically managed fields starting from the first yield trials at the latest. The project results were communicated to farmers and advisors during field days and excursions. They will be applied in the breeding of new cultivars

Skeletal Muscle–Specific Deletion of Lipoprotein Lipase Enhances Insulin Signaling in Skeletal Muscle but Causes Insulin Resistance in Liver and Other Tissues

OBJECTIVE—Skeletal muscle–specific LPL knockout mouse (SMLPL−/−) were created to study the systemic impact of reduced lipoprotein lipid delivery in skeletal muscle on insulin sensitivity, body weight, and composition

Chloramphenicol Selection of IS10 Transposition in the cat Promoter Region of Widely Used Cloning Vectors

The widely used pSU8 family of cloning vectors is based on a p15A replicon and a chloramphenicol acetyltransferase (cat) gene conferring chloramphenicol resistance. We frequently observed an increase in the size of plasmids derived from these vectors. Analysis of the bigger molecular species shows that they have an IS10 copy inserted at a specific site between the promoter and the cat open reading frame. Promoter activity from both ends of IS10 has been reported, suggesting that the insertion events could lead to higher CAT production. Insertions were observed in certain constructions containing inserts that could lead to plasmid instability. To test the possibility that IS10 insertions were selected as a response to chloramphenicol selection, we have grown these constructs in the presence of different amounts of antibiotic and we observed that insertions arise promptly under higher chloramphenicol selective pressure. IS10 is present in many E. coli laboratory strains, so the possibility of insertion in constructions involving cat-containing vectors should be taken into account. Using lower chloramphenicol concentrations could solve this problem

Why High-Performance Modelling and Simulation for Big Data Applications Matters

Modelling and Simulation (M&S) offer adequate abstractions to manage the complexity of analysing big data in scientific and engineering domains. Unfortunately, big data problems are often not easily amenable to efficient and effective use of High Performance Computing (HPC) facilities and technologies. Furthermore, M&S communities typically lack the detailed expertise required to exploit the full potential of HPC solutions while HPC specialists may not be fully aware of specific modelling and simulation requirements and applications. The COST Action IC1406 High-Performance Modelling and Simulation for Big Data Applications has created a strategic framework to foster interaction between M&S experts from various application domains on the one hand and HPC experts on the other hand to develop effective solutions for big data applications. One of the tangible outcomes of the COST Action is a collection of case studies from various computing domains. Each case study brought together both HPC and M&S experts, giving witness of the effective cross-pollination facilitated by the COST Action. In this introductory article we argue why joining forces between M&S and HPC communities is both timely in the big data era and crucial for success in many application domains. Moreover, we provide an overview on the state of the art in the various research areas concerned