Biomedical subjects

Silke Wandschneider

Publications and source records attributed to Silke Wandschneider.

4 recordsLinked to original sources

PubMed2005

Proteome analysis of isolated myenteric plexus reveals significant changes in protein expression during postnatal development.

The enteric nervous system in vertebrates is the most complex part of the peripheral nervous system. Concerning chemical coding, ultrastructure and neuronal circuits, it is more similar to the central than to the peripheral nervous system. Its networks, the myenteric and submucous plexus are integrated in the gut wall. The enteric nervous system is a system of high plasticity, which not only changes during pre- and postnatal development, but also with disease or changing dietary habits. The Aim of this study was to elucidate changes in protein expression during the first two postnatal weeks in the rat myenteric plexus. Colonic and duodenal myenteric plexus from newborn (P1) and fourteen-day old (P14) Sprague-Dawley rats was isolated following a procedure that combines enzymatic digestion and mechanical agitation. The neuronal tissue was collected and processed for two-dimensional gel electrophoresis (2-DE). The obtained 2-D gels were stained with silver for image analysis or with colloidal Coomassie for subsequent protein identification. Gels from the various samples showed a high degree of consistence concerning protein-spots found in all preparations. Nevertheless, there was a number of proteins that were clearly detected in one sample but not, or only in significantly smaller amounts in the other. Several differentially expressed proteins in the postnatal myenteric plexus were identified with MALDI-TOF mass spectrometry. Especially stathmin, polyubiquitin and heterogeneous nuclear ribonucleoprotein seem to play an important role in pre- and postnatal development. 2-DE combined with mass spectrometry can help to identify pathological relevant proteins in the enteric nervous system, and so deliver a valuable tool for the early diagnosis of also central nervous system diseases by using biopsies from the gut.

Animals↗

PubMed2005

Large-scale protein expression for proteome research.

Access to pure and soluble recombinant proteins is essential for numerous applications in proteome research, such as the production of antibodies, structural characterization of proteins, and protein microarrays. Through the German cDNA Consortium we have access to more than 1500 ORFs encoding uncharacterized proteins. Preparing a large number of recombinant proteins calls for the careful refinement and re-evaluation of protein purification tools. The expression and purification strategy should result in mg quantities of protein that can be employed in microarray-based assays. In addition, the experimental set-up should be robust enough to allow both automated protein expression screening and the production of the proteins on a mg scale. These requirements are best fulfilled by a bacterial expression system such as Escherichia coli. To develop an efficient expression strategy, 75 different ORFs were transferred into suitable expression vectors using the Gateway cloning system. Four different fusion tags (E. coli transcription-termination anti-termination factor (NusA), hexahistidine tag (6xHis), maltose binding protein (MBP) and GST) were analyzed for their effect on yield of induced fusion protein and its solubility, as determined at two different induction temperatures. Affinity-purified fusion proteins were confirmed by MALDI-TOF MS.

Amino Acid Sequence↗

PubMed2005

Use of proteomics to study chemosensitivity.

Chemoresistance remains an unresolved problem in clinical oncology. Therefore it is important to identify molecular factors that lead to an understanding of the mechanisms of drug resistance in cancer cells. On the protein-expression level, this can be done using proteomics, which has become the focus of significant interest and research over the past decade. We describe an easy and practicable standardized technique that can be used to study global protein expression in chemosensitive and chemoresistant cancer cells to find candidate proteins that are potentially associated with the drug-resistant phenotype. As an example, fractionation of human neuroblastoma cells using two-dimensional polyacrylamide electrophoresis, spot detection, image analysis, and finally protein identification is illustrated.

Antineoplastic Agents↗

PubMed2004

Prevention of cold-preservation injury of cultured endothelial cells by catecholamines and related compounds.

The present study was conducted to dissect the underlying mechanisms by which catecholamines protect cells against preservation injury. To this end, we firstly defined the cellular and molecular differences between protected and nonprotected cells and secondly defined the mediators that were involved in cold-induced damage. Cold storage of untreated human umbilical vein endothelial cells (HUVECs) resulted in profound cellular damage as assessed by lactate dehydrogenase (LDH) release and by morphological changes, e.g. cell size alterations and loss of cytoskeletal organization. Treatment of HUVECs with catecholamines before cold storage prevented cellular damage in a dose- and time-dependent fashion. Similar results were obtained with carvedilol or its hydroxylated derivative BM91.0228. Protection was not receptor-mediated and did not require de novo protein synthesis. The onset of protection occurred relatively quickly and the duration was long lasting. Addition of superoxide dismutase (SOD) to untreated HUVECs during cold preservation also was protective. Oxidation of catecholamines completely abrogated the protective effect of these compounds on cold-induced damage. Both at 4 degrees and 37 degrees C, catecholamines reduced the amount of reactive oxygen species (ROS) produced by HUVECs. In conclusion we have demonstrated that catecholamines protect cells against preservation injury either by scavenging of ROS or by inhibition of ROS production.

Blotting, Western↗