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1.
Lattenmayer C Loeschel M Schriebl K Steinfellner W Sterovsky T Trummer E Vorauer-Uhl K Müller D Katinger H Kunert R 《Biotechnology and bioengineering》2007,96(6):1118-1126
In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development. 相似文献
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Oettl K Payerl D Zitta S Müller T Estelberger W Reibnegger G 《Analytical biochemistry》2004,331(1):183-188
Sinistrin, as inulin, is widely used as a marker for renal function testing. A reliable and accurate method with a simple sample preparation for the quantitative determination of sinistrin would be of advantage. We developed a high-performance liquid chromatography (HPLC)-based method with electrochemical detection for the quantitative measurement of sinistrin in serum and plasma. Sample preparation is easy and includes enzymatic removal of glucose, deproteinization, acid hydrolysis of sinistrin, and HPLC separation of fructose. The recovery of sinistrin from serum is the same as that from water and is near 100%. The method presented has a linear range up to 500 mg/L. The results from our method are in agreement with a fully enzymatic quantification (regression coefficient 1.01, coefficient of variation 0.97). Sinistrin concentrations in aqueous solutions can be measured down to 2mg/L with a coefficient of variation of 5.7%. Quantification in serum is primarily limited by its physiological fructose content. The sensitivity of the described method is sufficient for its use in renal function testing. We describe a method for quantification of sinistrin which allows accurate measurements especially at low concentrations and low sample volumes. This laboratory method may be used for obtaining sinistrin pharmacokinetics in renal function testing. 相似文献
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Schliemann W Schneider B Wray V Schmidt J Nimtz M Porzel A Böhm H 《Phytochemistry》2006,67(2):191-201
From yellow petals of Iceland poppy, besides the known flavonoid gossypitrin, seven kaempferol derivatives were isolated. In addition to kaempferol 3-O-beta-sophoroside and kaempferol 3-O-beta-sophoroside-7-O-beta-glucoside, known from other plants, the mono- and dimalonyl conjugates of the latter were identified by MS and NMR spectroscopy. Structure analyses of a set of co-occurring pigments, the nudicaulins, revealed that they have the identical acylated glycoside moieties attached to a pentacyclic indole alkaloid skeleton for which the structure of 19-(4-hydroxyphenyl)-10H-1,10-ethenochromeno[2,3-b]indole-6,8,18-triol was deduced from MS and NMR as well as chemical and chiroptical methods. 相似文献
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Julian Willibald Stefanie Breukers Anjan Patel Christer L. Øpstad Stine Nalum Naess Vassilia Partali 《Chemistry and physics of lipids》2009,161(1):32-37
Stable cationic carotenoid aggregates — predominantly of the J-type — develop when the hydrochlorides of carotenoid aldoximes and ketoximes are exposed to water. The oxime hydrochlorides are obtained by simple syntheses from commercially available food color carotenoids. Bluish-purple, unstable transient compounds were observed during hydrochlorination performed at liquid nitrogen temperature. 相似文献
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Metabolite profiling of soluble primary and secondary metabolites, as well as cell wall-bound phenolic compounds from roots of barrel medic (Medicago truncatula) was carried out by GC-MS, HPLC and LC-MS. These analyses revealed a number of metabolic characteristics over 56 days of symbiotic interaction with the arbuscular mycorrhizal (AM) fungus Glomus intraradices, when compared to the controls, i.e. nonmycorrhizal roots supplied with low and high amounts of phosphate. During the most active stages of overall root mycorrhization, elevated levels of certain amino acids (Glu, Asp, Asn) were observed accompanied by increases in amounts of some fatty acids (palmitic and oleic acids), indicating a mycorrhiza-specific activation of plastidial metabolism. In addition, some accumulating fungus-specific fatty acids (palmitvaccenic and vaccenic acids) were assigned that may be used as markers of fungal root colonization. Stimulation of the biosynthesis of some constitutive isoflavonoids (daidzein, ononin and malonylononin) occurred, however, only at late stages of root mycorrhization. Increase of the levels of saponins correlated AM-independently with plant growth. Only in AM roots was the accumulation of apocarotenoids (cyclohexenone and mycorradicin derivatives) observed. The structures of the unknown cyclohexenone derivatives were identified by spectroscopic methods as glucosides of blumenol C and 13-hydroxyblumenol C and their corresponding malonyl conjugates. During mycorrhization, the levels of typical cell wall-bound phenolics (e.g. 4-hydroxybenzaldehyde, vanillin, ferulic acid) did not change; however, high amounts of cell wall-bound tyrosol were exclusively detected in AM roots. Principal component analyses of nonpolar primary and secondary metabolites clearly separated AM roots from those of the controls, which was confirmed by an hierarchical cluster analysis. Circular networks of primary nonpolar metabolites showed stronger and more frequent correlations between metabolites in the mycorrhizal roots. The same trend, but to a lesser extent, was observed in nonmycorrhizal roots supplied with high amounts of phosphate. These results indicate a tighter control of primary metabolism in AM roots compared to control plants. Network correlation analyses revealed distinct clusters of amino acids and sugars/aliphatic acids with strong metabolic correlations among one another in all plants analyzed; however, mycorrhizal symbiosis reduced the cluster separation and enlarged the sugar cluster size. The amino acid clusters represent groups of metabolites with strong correlations among one another (cliques) that are differently composed in mycorrhizal and nonmycorrhizal roots. In conclusion, the present work shows for the first time that there are clear differences in development- and symbiosis-dependent primary and secondary metabolism of M. truncatula roots. 相似文献
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Hannes Reisinger Willibald Steinfellner Hermann Katinger Renate Kunert 《Cytotechnology》2009,60(1-3):115-123
The generation of transgenic cell lines is acquired by facilitating the uptake and integration of DNA. Unfortunately, most of the systems generating stable expression systems are cost and time-consuming and transient expression is optimized to generate milligram amounts of the recombinant protein. Therefore we improved and compared two transfection systems, one based on cationic liposomes consisting of DOTAP/DOPE and the second one on polyethylenimine (PEI). Both systems have been used as chemically defined transfection systems in combination with serum-free cultivated host cell line. At first we had determined the toxicity and ideal ratio of DNA to PEI followed by determination of the optimal transfection conditions in order to achieve maximum transfection efficiency. We then directly compared DOTAP/DOPE and PEI in transient transfection experiments using enhanced green fluorescence protein (EGFP) and a human monoclonal antibody, mAb 2F5, as a model protein. The results which were achieved in case of EGFP were more than 15% transfectants at a viability of 85%. Despite the fact that expression of the mAb was found negligible we used both techniques to generate stable mAb 2F5 expressing cell lines that underwent several cycles of screening and amplification with methotrexate, and resulted in cell lines with similar volumetric production titers. These experiments serve to demonstrate the potential of stable cell lines even in case where the transient systems did not show satisfying results. 相似文献
8.
Franziska Gro?sch?dl Wolfgang Freidl éva Rásky Nathalie Burkert Johanna Muckenhuber Willibald J. Stronegger 《PloS one》2014,9(9)
Background
The prevalence of back pain is constantly increasing and a public health problem of high priority. In Austria there is a lack of empirical evidence for the development of back pain and its related factors. The present study aims to investigate trends in the prevalence of back pain across different subpopulations (sex, age, obesity).Methods
A secondary data analysis based on five nationally representative cross-sectional health surveys (1973–2007) was carried out. Face-to-face interviews were conducted in private homes in Austria. Subjects aged 20 years and older were included in the study sample (n = 178,818). Obesity was defined as BMI≥30 kg/m2 and adjusted for self-report bias. Back pain was measured as the self-reported presence of the disorder.Results
The age-standardized prevalence of back pain was 32.9% in 2007; it was higher among women than men (p<0.001), higher in older than younger subjects (p<0.001) and higher in obese than non-obese individuals (p<0.001). During the investigation period the absolute change in the prevalence of back pain was +19.4%. Among all subpopulations the prevalence steadily increased. Obese men showed the highest increase of and the greatest risk for back pain.Conclusion
These results help to understand the development of back pain in Austria and can be used to plan controlled promotion programs. Further monitoring is recommended in order to control risk groups and plan target group-specific prevention strategies. In Austria particular emphasis should be on obese individuals. We recommend conducting prospective studies to confirm our results and investigate causal relationships. 相似文献9.
Ana Stevanovic Mark Coburn Ares Menon Rolf Rossaint Daren Heyland Gereon Sch?lte Thilo Werker Willibald Wonisch Michael Kiehntopf Andreas Goetzenich Steffen Rex Christian Stoppe 《PloS one》2014,9(8)
Introduction
Cardiac surgery is accompanied by an increase of oxidative stress, a significantly reduced antioxidant (AOX) capacity, postoperative inflammation, all of which may promote the development of organ dysfunction and an increase in mortality. Selenium is an essential co-factor of various antioxidant enzymes. We hypothesized a less pronounced decrease of circulating selenium levels in patients undergoing off-pump coronary artery bypass (OPCAB) surgery due to less intraoperative oxidative stress.Methods
In this prospective randomised, interventional trial, 40 patients scheduled for elective coronary artery bypass grafting were randomly assigned to undergo either on-pump or OPCAB-surgery, if both techniques were feasible for the single patient. Clinical data, myocardial damage assessed by myocard specific creatine kinase isoenzyme (CK-MB), circulating whole blood levels of selenium, oxidative stress assessed by asymmetric dimethylarginine (ADMA) levels, antioxidant capacity determined by glutathionperoxidase (GPx) levels and perioperative inflammation represented by interleukin-6 (IL-6) levels were measured at predefined perioperative time points.Results
At end of surgery, both groups showed a comparable decrease of circulating selenium concentrations. Likewise, levels of oxidative stress and IL-6 were comparable in both groups. Selenium levels correlated with antioxidant capacity (GPx: r = 0.720; p<0.001) and showed a negative correlation to myocardial damage (CK-MB: r = −0.571, p<0.001). Low postoperative selenium levels had a high predictive value for the occurrence of any postoperative complication.Conclusions
OPCAB surgery is not associated with less oxidative stress and a better preservation of the circulating selenium pool than on-pump surgery. Low postoperative selenium levels are predictive for the development of complications.Trial registration
ClinicalTrials.gov NCT01409057相似文献10.