Determination of Ochratoxin A in small volumes of human blood serum

A new simple and rapid method for analysing Ochratoxin A (OTA) in small volumes of human blood serum using capillary zone electrophoresis coupled to laser-induced fluorescence is described. The clean-up procedure solely consists of a double extraction step. To improve the reproducibility of migration times and quantification, two internal standards were used. The limit of detection was 0.55 ng/ml, with a linear range of 1-100 ng/ml of OTA in spiked human blood serum. The method is used to rapidly screen suspected patients.     

Affinity parameters of amino acid derivative binding to molecularly imprinted nanospheres consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)

The binding of L-Boc-phenylalanine anilide (BFA) and L-Boc-phenylalanine (phe) to molecularly imprinted and non-imprinted polymer nanoparticles consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)] has been investigated by adsorption experiments and mathematical modeling. The experimental isotherms have been mathematically adapted following the models of Freundlich, Langmuir, Langmuir-Freundlich, Bi-Langmuir, and extended Langmuir. The extended Langmuir model differentiated between specific and nonspecific binding of the ligand to the receptor nanoparticles and rendered excellent fitting of the experimental data. It delivered a thermodynamic and kinetic parameter set on the experimental association curves of L-BFA by L-BFA-imprinted nanospheres in suspension experiments with the equilibrium constant KD= 4.09 +/- 0.69 micromol L(-1) and the kinetic association rate constant Ka= 5.60 mL micromol(-1) min(-1).     

Flexible automation of cell culture and tissue engineering tasks

Until now, the predominant use cases of industrial robots have been routine handling tasks in the automotive industry. In biotechnology and tissue engineering, in contrast, only very few tasks have been automated with robots. New developments in robot platform and robot sensor technology, however, make it possible to automate plants that largely depend on human interaction with the production process, e.g., for material and cell culture fluid handling, transportation, operation of equipment, and maintenance. In this paper we present a robot system that lends itself to automating routine tasks in biotechnology but also has the potential to automate other production facilities that are similar in process structure. After motivating the design goals, we describe the system and its operation, illustrate sample runs, and give an assessment of the advantages. We conclude this paper by giving an outlook on possible further developments.     

Human molecular chronotyping in sight?

Recent research on mouse models has taken us closer to deciphering the molecular clock mechanism that defines an individual's 'body time'. How feasible will it be to create a molecular timetable that allows determination of individual body time from tissue harvested at a single time point?     

<Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> type III secretion: Evidence for the ability to transport proteins that are folded prior to secretion

Background  

Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis) share a type three secretion system (TTSS) which allows translocation of effector proteins (called Yops) into host cells. It is believed that proteins are delivered through a hollow needle with an inner diameter of 2–3 nm. Thus transport seems to require substrates which are essentially unfolded. Recent work from different groups suggests that the Yersinia TTSS cannot accommodate substrates which are folded prior to secretion. It was suggested that folding is prevented either by co-translational secretion or by the assistance of specific Yop chaperones (called Sycs).     

Repression of primordial germ cell differentiation parallels germ line stem cell maintenance

In Drosophila, primordial germ cells (PGCs) are set aside from somatic cells and subsequently migrate through the embryo and associate with somatic gonadal cells to form the embryonic gonad. During larval stages, PGCs proliferate in the female gonad, and a subset of PGCs are selected at late larval stages to become germ line stem cells (GSCs), the source of continuous egg production throughout adulthood. However, the degree of similarity between PGCs and the self-renewing GSCs is unclear. Here we show that many of the genes that are required for GSC maintenance in adults are also required to prevent precocious differentiation of PGCs within the larval ovary. We show that following overexpression of the GSC-differentiation gene bag of marbles (bam), PGCs differentiate to form cysts without becoming GSCs. Furthermore, PGCs that are mutant for nanos (nos), pumilio (pum) or for signaling components of the decapentaplegic (dpp) pathway also differentiate. The similarity in the genes necessary for GSC maintenance and the repression of PGC differentiation suggest that PGCs and GSCs may be functionally equivalent and that the larval gonad functions as a "PGC niche".     

WW domain sequence activity relationships identified using ligand recognition propensities of 42 WW domains

WW domains mediate protein-protein interactions in a number of different cellular functions by recognizing proline-containing peptide sequences. We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens. As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues. Thirty-two WW domains were classified into six groups according to detected ligand recognition preferences for binding the motifs PPx(Y/poY), (p/phi)P(p,g)PPpR, (p/phi)PPRgpPp, PPLPp, (p/xi)PPPPP, and (poS/poT)P (motifs according to modified Seefeld Convention 2001). In addition to these distinct binding motifs, group-specific WW domain consensus sequences were identified. For PPxY-recognizing domains, phospho-tyrosine binding was also observed. Based on the sequences of the PPx(Y/poY)-specific group, a profile hidden Markov model was calculated and used to predict PPx(Y/poY)-recognition activity for WW domains, which were not assayed. PPx(Y/poY)-binding was found to be a common property of NEDD4-like ubiquitin ligases.     

Vasopressin type 1A receptor up-regulation by cyclosporin A in vascular smooth muscle cells is mediated by superoxide

Based on our previous results, we investigated whether cyclosporin A (CsA)-induced vasopressin type 1A receptor up-regulation was mediated by free radicals. We report that CsA analogues with different affinities for cyclophilin and calcineurin were able to up-regulate vasopressin type 1A receptor and to generate free radicals in smooth muscle cells independently of calcineurin. Further, we demonstrate that the antioxidant N-acetyl-L-cysteine blocked the increase in vasopressin type 1A receptor mRNA and protein levels induced by CsA and that low concentrations of prooxidants were able to directly increase vasopressin type 1A receptor mRNA and protein levels. In addition, short exposure to CsA or pro-oxidants was sufficient to significantly increase vasopressin type 1A receptor mRNA and protein levels. Using cell-permeable forms of superoxide dismutase and catalase, we finally show that superoxide mediates the CsA-induced effects on vasopressin type 1A receptor. These results provide strong evidence that CsA-induced superoxide generation is causally involved in vasopressin type 1A receptor expression and demonstrate for the first time that low physiological concentrations of radicals, most probably superoxide, are able to directly affect cellular signaling to increase vasopressin type 1A receptor expression in rat aortic smooth muscle cells.     

Stable isotope phospho-profiling of fibrinogen and fetuin subunits by element mass spectrometry coupled to capillary liquid chromatography

We have used one-dimensional polyacrylamide gel electrophoresis, tryptic digestion, and capillary liquid chromatography-mass spectrometry with inductively coupled plasma ionization and phosphorus-31 detection or electrospray ionization for the analysis of protein phosphorylation. We have analyzed human fibrinogen with two well-characterized phosphorylation sites and bovine fetuin with unknown phosphorylation status. Both serine-3 and serine-345 (both in Aalpha) of fibrinogen were clearly recognized. In bovine fetuin, four phosphorylated sites were newly characterized (serine-138, serine-320, serine-323, and serine-324). The novel strategy provides a fast and quantitative overview of the presence of protein phosphorylation sites.