Identification of microRNAs specific for high producer CHO cell lines using steady-state cultivation

MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene expression. Hence, microRNAs are considered as potential targets for engineering of Chinese hamster ovary (CHO) cells to improve recombinant protein production. Here, we analyzed and compared the microRNA expression patterns of high, low, and non-producing recombinant CHO cell lines expressing two structurally different model proteins in order to identify microRNAs that are involved in heterologous protein synthesis and secretion and thus might be promising targets for cell engineering to increase productivity. To generate reproducible and comparable data, the cells were cultivated in a bioreactor under steady-state conditions. Global microRNA expression analysis showed that mature microRNAs were predominantly upregulated in the producing cell lines compared to the non-producer. Several microRNAs were significantly differentially expressed between high and low producers, but none of them commonly for both model proteins. The identification of target messenger RNAs (mRNAs) is essential to understand the biological function of microRNAs. Therefore, we negatively correlated microRNA and global mRNA expression data and combined them with computationally predicted and experimentally validated targets. However, statistical analysis of the identified microRNA-mRNA interactions indicated a considerable false positive rate. Our results and the comparison to published data suggest that the reaction of CHO cells to the heterologous protein expression is strongly product- and/or clone-specific. In addition, this study highlights the urgent need for reliable CHO-specific microRNA target prediction tools and experimentally validated target databases in order to facilitate functional analysis of high-throughput microRNA expression data in CHO cells.     

Genetic recombination events which position the friedreich ataxia locus proximal to the D9S15/D9S5 linkage group on chromosome 9q

The absence of recombination between the mutation causing Friedreich ataxia and the two loci which originally assigned the disease locus to chromosome 9 has slowed attempts to isolate and characterize the genetic defect underlying this neurodegenerative disorder. A proximity of less than 1 cM to the linkage group has been proved by the generation of high maximal lod score (Z) to each of the two tightly linked markers D9S15 (Z = 96.69; recombination fraction [θ] = .01) and D9S5 (Z = 98.22; θ = .01). We report here recombination events which indicate that the FRDA locus is located centromeric to the D9S15/D9S5 linkage group, with the most probable order being cen–FRDA–D9S5–D9S15–qter. However, orientation of the markers with respect to the centromere, critical to the positional cloning strategy, remains to be resolved definitively.     

Peptide Bβ15-42 Preserves Endothelial Barrier Function in Shock

Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bß15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bß15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bß15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bß15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bß15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bß15-42 is confirmed in Fyn^−/− mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bß15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock.     

The regeneration of neuromuscular junctions during spontaneous re-innervation of the rat diaphragm

Summary Electron microscopic observations have been made on the regeneration of neuromuscular junctions during spontaneous re-innervation of the rat diaphragm, following unilateral transsection of the phrenic nerve. 3 and 4 weeks after denervation motor end plates displayed the pattern of almost complete degeneration, i.e. persisting subneural foldings, deprived of neural contact and covered with collagen fibrils and fibrocytes. From observations at 5, 10 and 24 weeks after denervation the following sequence of events could be established: a few small axon terminals, accompanied by Schwann cells, became apposed to subneural folds, while most foldings were covered initially by Schwann cells or still by collagen fibrils. Gradually an increasing number of subneural folds came into contact with axon terminals. At 24 weeks all junctions displayed the pattern of a mature motor end plate. In the majority of regenerating neuromuscular junctions single dense-cored vesicles of approximately 900–1200 Å were present in axon terminals.It is concluded that under the present conditions restoration of neuromuscular transmission is accomplished by a re-innervation of the preserved subneural apparatuses of former junctions by regenerating axons. The significance of the occurrence of dense-cored vesicles in regenerating motor end plates is discussed.This work was supported by the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk.     

On the ultrastructure of the rat anococcygeus muscle

Cell and Tissue Research - The rat anococcygeus muscle, which is known from previous functional and histochemical investigations to be a smooth muscle with a dense adrenergic innervation, was...     

A new method to measure nitrate/nitrite with a NO-sensitive electrode

There are different methods to measure the unstable moleculenitric oxide (NO). We will describe a new sensitive method to measure NO by reconversion of nitrate/nitrite to NO, which will bedetermined with an amperometric Clark-type electrode. Nitrate andnitrite are the degradation products of NO. First, nitrate isenzymatically converted to nitrite with the use of the nitrate reductase. Second, nitrite is reduced to equimolar NO concentrations byan acidic iodide solution. The detection limit of the electrode in anaqueous solution was 2 nmol/l NO (meaning the threshold was dependingon the volume added: 500 µl of a 0.2 µmol/l nitrite solution addedto a 10-ml bath). This method provides the ability to assess basal andagonist-stimulated NO releases of different biological models. Wemeasured basal and carbachol-stimulated NO release of nativeendothelial cells from porcine coronary arteries and porcine aorticendothelial cell cultures. Moreover, it was possible to measure thenitrate/nitrite concentration in the coronary effluent of a guinea pigheart. In conclusion, we present a valid, highly sensitive new methodof measuring nitrite/NO in biological systems with a commerciallyavailable electrode.

     

Aufenthaltsdauern explorierender Meisen: Habitat- und Artspezifit?t

Ohne Zusammenfassung

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Patch residence times of exploring tits: habitat and species specificity

     

Frequent diploidisation of haploid Armillaria ostoyae strains in an outdoor inoculation experiment

Very little is known about the biology and ecology of haploid Armillaria strains in nature. In this outdoor inoculation experiment, we assessed the virulence of six haploid Armillariaostoyae strains along with their diploid parent towards 2-year-old seedlings and 4-year-old saplings of Norway spruce (Picea abies), and determined their ability to colonise freshly cut stumps. As inoculum source an Armillaria-colonised hazelnut (Corylus avellana) stem segment was inserted into the soil substrate. Re-isolations from mycelial fans at the root collar of infected trees or stumps were made. Surprisingly, not a single haploid re-isolate could be recovered. Microsatellite genotyping of 133 re-isolates suggests that the inoculated haploid strains were diploidised either by mating propagules (basidiospores or haploid mycelia) already present in the soil substrate or naturally disseminated in the course of the experiment from nearby forests. Consequently, no conclusion about the infectious ability of haploid Armillaria mycelia under natural conditions can be drawn. Nonetheless, the diploid half-sib families resulting from the diploidisation showed varying degrees of virulence, with a high correlation between the experiment with 2-year-old seedlings and 4-year-old saplings. Despite extensive genotyping of re-isolates, no evidence for somatic recombination between haploid mating propagules and diploidised mycelia was detected, suggesting that this is an uncommon phenomenon in A. ostoyae.