Severe hypomyelination of the murine CNS in the absence of myelin-associated glycoprotein and fyn tyrosine kinase.

The analysis of mice deficient in the myelin-associated glycoprotein (MAG) or Fyn, a nonreceptor-type tyrosine kinase proposed to act as a signaling molecule downstream of MAG, has revealed that both molecules are involved in the initiation of myelination. To obtain more insights into the role of the MAG-Fyn signaling pathway during initiation of myelination and formation of morphologically intact myelin sheaths, we have analyzed optic nerves of MAG-, Fyn- and MAG/Fyn-deficient mice. We observed a slight hypomyelination in optic nerves of MAG mutants that was significantly increased in Fyn mutants and massive in MAG/Fyn double mutants. The severe morphological phenotype of MAG/Fyn mutants, accompanied by behavioral deficits, substantiates the importance of both molecules for the initiation of myelination. The different severity of the phenotype of different genotypes indicates that the MAG-Fyn signaling pathway is complex and suggests the presence of compensatory mechanisms in the single mutants. However, data are also compatible with the possibility that MAG and Fyn act independently to initiate myelination. Hypomyelination of optic nerves was not related to a loss of oligodendrocytes, indicating that the phenotype results from impaired interactions between oligodendrocyte processes and axons and/or impaired morphological maturation of oligodendrocytes. Finally, we demonstrate that Fyn, unlike MAG, is not involved in the formation of ultrastructurally intact myelin sheaths.

Application of Prionics Western blotting procedure to screen for BSE in cattle regularly slaughtered at Swiss abattoirs.

Disease-specific PrP (PrP(Sc)) is at least part of the infectious particle (prion) causing bovine spongiform encephalopathy (BSE) or scrapie in sheep. Digestion with protease allows a distinction between normal PrP (PrP(C)) and PrP(Sc) i.e. PrP(C) is completely digested while PrP(Sc) is cleaved at the N-terminus leading to a fragment of reduced molecular weight (PrP 27-30). Detection of this fragment by Western blotting has been described more than a decade ago for rodent PrP. We have now optimized the technique in order to allow rapid analysis of hundreds of samples per day. Here we report the application of this technique to the analysis of 3000 regularly slaughtered cattle from Swiss abattoirs. For comparison all the animals were subsequently examined by classical methods (i.e. histology and immunohistochemistry). All but one animal were negative for BSE by all methods. The Western blot positive animal was confirmed to be a BSE case and the carcass was removed from the food chain. We conclude that it is feasible to examine slaughtered cattle on a routine basis without causing delays to the meat processing industry.

Validation of a western immunoblotting procedure for bovine PrP(Sc) detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE).

In this report we document the results of several independent studies testing the sensitivity, specificity and reliability of the Prionics Western blotting (PWB) procedure to detect bovine and ovine disease-specific, protease-resistant prion protein (PrP(Sc)). Validation of the technique was obtained by blind analysis of samples from cattle affected with bovine spongiform encephalopathy (BSE), clinically normal animals or cattle with neurological diseases unrelated to BSE. Overall, very high sensitivity, specificity and reliability was observed. It became clear that sampling of the correct brain region and the method used for protein extraction are important factors for correct diagnosis. Furthermore, we tested the usefulness of the PWB technique as an instrument for surveillance purposes. We analyzed animals from a culling scheme as well as older animals from abattoirs to determine the number of subclinical BSE cases detectable by histopathological examination, immunohistochemistry for PrP(Sc) and PWB. In both studies, BSE-affected animals with no overt clinical symptoms were detected. These results demonstrate the usefulness of the PWB procedure in surveillance systems serving as a rapid diagnostic tool to identify animals subclinically infected with BSE.