Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol

We present a fast protocol that can be used to obtain highly purified cultures of proliferating adult human and rat Schwann cells accessible for non-viral transfection methods. The use of enriched genetically modified adult Schwann cells is of interest in the context of autologous cell transplantation within nerve transplants for peripheral nerve repair. Cell preparation from pre-degenerated adult peripheral nerves is described, together with the use of melanocyte growth medium plus forskolin, fibroblast growth factor-2 (FGF-2), pituitary extract and heregulin as a selective, serum-free culture medium and a subsequent cell enrichment step (cold jet). Proliferating adult Schwann cells can be efficiently genetically modified using optimized, non-viral electroporation protocols. The protocol results in Schwann cell cultures that are more than 90-95% pure, and transfection efficiencies vary depending on the initial cell constitution from 20 to 40%. The procedure takes up to 21 d, depending on the length of the pre-degeneration period.     

Subspace projection approaches to classification and visualization of neural network-level encoding patterns

Recent advances in large-scale ensemble recordings allow monitoring of activity patterns of several hundreds of neurons in freely behaving animals. The emergence of such high-dimensional datasets poses challenges for the identification and analysis of dynamical network patterns. While several types of multivariate statistical methods have been used for integrating responses from multiple neurons, their effectiveness in pattern classification and predictive power has not been compared in a direct and systematic manner. Here we systematically employed a series of projection methods, such as Multiple Discriminant Analysis (MDA), Principal Components Analysis (PCA) and Artificial Neural Networks (ANN), and compared them with non-projection multivariate statistical methods such as Multivariate Gaussian Distributions (MGD). Our analyses of hippocampal data recorded during episodic memory events and cortical data simulated during face perception or arm movements illustrate how low-dimensional encoding subspaces can reveal the existence of network-level ensemble representations. We show how the use of regularization methods can prevent these statistical methods from over-fitting of training data sets when the trial numbers are much smaller than the number of recorded units. Moreover, we investigated the extent to which the computations implemented by the projection methods reflect the underlying hierarchical properties of the neural populations. Based on their ability to extract the essential features for pattern classification, we conclude that the typical performance ranking of these methods on under-sampled neural data of large dimension is MDA>PCA>ANN>MGD.     

Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K(m)). The enzyme may play a role in lignocellulose degradation.     

Development of an HPLC method for the determination of doripenem in human and mouse serum

A HPLC method utilizing solid phase extraction was developed to analyze doripenem (formerly S-4661) in human and mouse serum. A reversed-phase column was used with a UV detector set at 295 nm. The mobile phase consisted of methanol and phosphate buffer at a flow rate of 1.5 ml/min. Meropenem was used as the internal standard. The standard curve was linear over a range of 0.5-40 microg/ml. The assay is simple, reproducible, and accurate and has been used successfully to analyze doripenem concentrations from a murine pharmacokinetic study.     

SPINE: a method for the rapid detection and analysis of protein-protein interactions in vivo

The detection and analysis of protein-protein interactions is one of the central tasks of proteomics in the postgenomic era. For this purpose, we present a procedure, the Strep-protein interaction experiment (SPINE) that combines the advantages of the Strep-tag protein purification system with those of reversible in vivo protein crosslinking by formaldehyde. Using two Bacillus subtilis regulator proteins, we demonstrate that this method is well suited to isolate protein complexes with high purity and virtually no background. Plasmids allowing the high-level expression of proteins carrying an N- or C-terminal Strep-tag in B. subtilis were constructed.     

Bone-marrow mononuclear cell therapy of severe ischemic heart failure

We describe cell therapy for severe ischemic heart failure using transendocardial injection of autologous bone-marrow-derived mononuclear cells. The treated patients had significantly less heart failure and angina, sustained significant improvement of pumping power, exercise capacity, cardiac muscle irrigation, and blood supply to the body. Electrical and mechanical mappings of the myocardium before and after the therapy, and anatomopathological examination of the myocardium of one of the patients that had deceased of a stroke eleven months after the treatment indicated sustained neoangiogenesis and improvement of activity and quantity of cardiomyocytes in the injected regions. Post-hoc analyses of injected cell phenotype and improvement of myocardial function indicate that presence of CD8+ and CD56+ cells does not correlate with good prognostics, suggesting a possibility of cell selection. For 'no-option' severe cardiac patients, significant benefits of cell therapy and absence of adverse effects may justify the application of bone-marrow-derived cell therapy.