Substitutions of hydrophobic amino acids reduce the amyloidogenicity of Alzheimer's disease beta A4 peptides.

The deposition of amyloid protein aggregates in brain is the main pathological feature of Alzheimer's disease. Their principal constituent is a peptide termed beta A4, which comprises up to 43 amino acid residues. It is highly insoluble under physiological conditions and aggregates into filaments that form very dense clusters in vivo and in vitro. Based on a beta A4 prototype sequence spanning residues 10 to 42 or 43, we have designed analogues in which hydrophobic amino acid residues in position 17 to 20 were substituted by more hydrophilic residues. Depending on the kind of newly introduced amino acids and their position within the sequence, the substitution of only two residues led to variants exhibiting a broad spectrum of different properties. Common to them was a reduced beta-sheet content after solubilization in water and in the solid state. Some of the variants showed significantly reduced amyloidogenicity: although still forming filaments, they did not aggregate into the highly condensed depositions that are typical for amyloid. In addition, they could be solubilized in 200 mM-NaCl and KCl. When mixed with beta A4 peptides bearing the natural sequence, two of the analogues could inhibit the formation of filaments in vitro. These results demonstrate that a well-preserved hydrophobic core around residues 17 to 20 of beta A4 is crucial for the formation of beta-sheet structure and the amyloid properties of beta A4. The introduction of structural alterations within this region may guide the development of reagents for the therapy of Alzheimer's disease.     

Dual regulation of the yeast CDC28-p40 protein kinase complex: cell cycle, pheromone, and nutrient limitation effects

A 40 kd polypeptide that coprecipitates with the CDC28 gene product in immune complexes is specifically phosphorylated by the CDC28 protein kinase. Using this reaction, we detect activity only in extracts from dividing G1 phase cells. Exit from G1 by entry into S phase or the preconjugatory state induced by mating pheromone correlates with loss of p40 phosphorylation activity. Inactive extracts from cdc28 mutants complement extracts from cells arrested in S or M phase, suggesting that non-G1 cells are deficient in an exchangeable activating factor. Stationary and pheromone-treated cultures are rich in this exchangeable factor, but possess an inactive kinase that is not activated by complementation. cAMP-deficient mutants resemble stationary cells.     

Peripheral oestrogen metabolism in postmenopausal women with or without breast cancer: the role of dietary lipids and growth factors

Studies we have carried out have revealed significant differences in oestrogen production and metabolism between normal women and postmenopausal women with breast cancer. The free, biologically available fraction of oestradiol is elevated in plasma from women with breast cancer and we have found that metabolic clearance rates and production rates of oestradiol are also increased. In vitro studies have suggested that lipids can influence the distribution of sex steroids in plasma and we have therefore examined the effect of dietary lipids on the distribution of sex steroids in plasma in vivo. Consumption of a meal with a high saturated fat content or the oral or i.v. administration of "Intralipid", a stabilised emulsion of soya bean oil that is high in unsaturated free fatty acids, had little effect on the available fractions of oestradiol in plasma. However, results from a preliminary study suggest that long-term changes in dietary fat intake can alter the distribution of steroids in plasma. It is concluded that dietary lipids may influence the availability of sex steroids to tissues. Such a mechanism could account for the significant correlation that has been found between dietary fat consumption and the incidence of breast cancer on a world-wide basis.     

A New Non-Mendelian Genetic Element of Yeast That Increases Cytopathology Produced by M1 Double-Stranded RNA in ski Strains

The Saccharomyces cerevisiae SKI (superkiller) genes are repressors of replication of M, L-A, and L-BC double-stranded (ds) RNAs; ski strains have an increased M dsRNA copy number and, as a result, are cold-sensitive for growth at 8 degrees. Growth is normal, however, at higher temperatures. We have found a new cytoplasmic genetic element [D] (for disease) that makes M1 dsRNA-containing superkiller strains grow slowly at 30 degrees, not at all at 37 degrees, and only very poorly at 20 degrees. These growth defects require three factors: a chromosomal ski mutation, the presence of M1 dsRNA, and the presence of the new cytoplasmic factor, [D]. We have isolated mutants unable to maintain [D] (mad), at least one of which is due to mutation of a single chromosomal locus. Further, [D] can be cured by growth at 37-39 degrees. We present evidence that [D] is not M, L-A, L-BC or W dsRNAs or mitochondrial DNA, 2 mu DNA, or [psi], but [D] depends on L-A for its maintenance. We also show that [D] is distinct from [B], a cytoplasmic element that allows M1 dsRNA to be stably replicated and maintained in spite of defects in certain chromosomal MAK genes that would otherwise be necessary. [D] activity is blocked by the presence of another extrachromosomal element, called [DIN] (for [D] interference). [D] and [DIN] may be different natural variants of the same molecule.