Methodological aspects of rat beta-endorphin analysis-influence of diurnal variation.

Beta-endorphin radioimmunoassays (RIAs) are widely performed following physical, emotional and environmental challenges in the rat. In the literature, a wide range of techniques have been described, but in the present study, we have focused on methodological aspects of beta-endorphin RIAs, investigating various characteristics of human and rat specific antibodies. Initial studies verified that the RIA outcome was not appropriate when using non-species compatible components. Novel rat beta-endorphin antibodies, r 4114 and r 4268, were raised in rabbits and characterised in terms of specificity, avidity and titer. Both of the new antisera showed 68.1% cross-reactivity with human beta-endorphin. The ED50 was 50+/-8 pmol/l, and the mean ED80 was 17 pmol/l for r 4268 but three-fold higher for r 4114. The intra-assay coefficient of variation (CV) was 7% at 100 pmol/l and the inter-assay CV was 10% at the same level for r 4268 and similar for r 4114. Using this novel rat beta-endorphin RIA for analyses of diurnal influence and removal from the Animal House cage, no significant changes were observed in either the hypothalamus or peri-aqueductal grey regions. These results suggest that rat beta-endorphin concentrations in these brain areas are not affected by order of removal or diurnal variation.

Genetic association to the amyloid plaque associated protein gene COL25A1 in Alzheimer's disease.

The COL25A1 gene, located in 4q25, encodes the CLAC protein, which has been implicated in Alzheimer's disease (AD) pathogenesis. CLAC was originally identified in amyloid preparations from AD brain and has been shown to be associated with amyloid plaques, inhibition of Abeta-fibril elongation and increased protease resistance of Abeta-fibrils through direct binding to Abeta. These biochemical data as well as the genomic location of the COL25A1 gene in chromosome 4q25 where we previously have reported a weak linkage-signal in Swedish AD families encouraged us to perform a case-control association study of two LD blocks in COL25A1 using 817 AD cases and 364 controls. The LD blocks cover a putative Abeta-binding motif and the variable 3' end of the gene. The analyses indicated association to three of eight analysed SNPs. We found further support for the association by replication in a Swedish population-based longitudinal sample set (n=926). Thus, in addition to the biochemical data, there is now genetic evidence of association between COL25A1 and risk for Alzheimer's disease.