Formation of cytotoxic transthyretin is not dependent on inter-molecular disulphide bridges commonly found within the amyloid form.

Familial amyloidotic polyneuropathy (FAP) is linked to destabilising point mutations in the human plasma protein transthyretin (TTR). Consistent with similar amyloid disorders, low molecular weight TTR oligomers have been shown to exert the major cytotoxic effect. The amyloid structure of TTR contains non-native inter-molecular disulphide linkages via the cysteine at position 10 (Cys10). Moreover, substitution of Cys10 in a mouse model for TTR-amyloidosis abolishes TTR deposits, indicating an important role of Cys10 in FAP pathogenesis. However, the role of disulphide bridges in TTR cytotoxicity has not been elucidated. By probing Cys10Ser TTR variants to the human neuroblastoma SH-SY5Y cell line, we have addressed this question, and our results clearly show that formation of an inter-molecular disulphide bridge is not a pre-requisite for TTR cytotoxicity. This finding suggests that prevention of inter-molecular TTR disulphide bridges as a therapeutic intervention will not impair the cytotoxic potential of TTR.

Inhibition of beta-amyloid-induced neurotoxicity by imidazopyridoindoles derived from a synthetic combinatorial library.

Alzheimer's disease is a progressive neurodegenerative disorder characterized by the deposit of amyloid fibrils in the brain that result from the self-aggregative polymerization of the beta-amyloid peptide (Abeta). Evidence of a direct correlation between the ability of Abeta to form stable aggregates in aqueous solution and its neurotoxicity has been reported. The cytotoxic effects of Abeta have been attributed to the aggregation properties of a domain corresponding to the peptide fragment Abeta25-35. In an effort to generate novel inhibitors of Abeta neurotoxicity and/or aggregation, a mixture-based synthetic combinatorial library composed of 23 375 imidazopyridoindoles was generated and screened for inhibition of Abeta25-35 neurotoxicity toward the rat pheochromocytoma PC-12 cell line. The effect of the identified lead compounds on Abeta25-35 aggregation was then evaluated by means of circular dichroism (CD) and thioflavin-T fluorescence spectroscopy. Their activity against Abeta1-42 neurotoxicity toward the PC-12 cell line was also determined. The most active imidazopyridoindoles inhibited both Abeta25-35 and Abeta1-42 neurotoxicity in the low- to mid-micromolar range. Furthermore, inhibition of the random coil to beta-sheet transition and self-aggregation of Abeta25-35 was observed by CD and fluorescence spectroscopy, supporting the relationship between inhibition of the Abeta aggregation process and neurotoxicity.

Biotransformations of putative phytoecdysteroid biosynthetic precursors in tissue cultures of Polypodium vulgare.

Incubation of calli and prothalli of Polypodium vulgare with different tritium-labelled ecdysteroids has led to modification of some previous assumptions about the biosynthesis of ecdysteroids in plants. Thus, 25-deoxy-20-hydroxyecdysone was transformed efficiently in both tissues into 20-hydroxyecdysone (20E), but no 25-deoxyecdysteroids such as pterosterone and inokosterone were formed. Likewise, incubation of 2-deoxyecdysone (2dE) produced exclusively ecdysone (E) and 20E, indicating a high 2-hydroxylase activity in both tissues, despite calli not producing phytoecdysteroids. This 2-hydroxylation was also evident in the transformation of 2,22-dideoxyecdysone (2,22dE) into 22-deoxyecdysone (22dE). Different ecdysteroids that do not occur in P. vulgare were formed in the incubation of 3-dehydro-2,22,25-trideoxyecdysone (3D2,22,25dE) by 3alpha-reduction and 3beta-reduction and 25-hydroxylation processes. The fact that 22,25-dideoxyecdysone and 22dE were the only 2-hydroxylated products formed in this case suggests that only compounds bearing a 3beta-hydroxyl group are substrates for the 2-hydroxylase. Surprisingly, 22-hydroxylation was never observed with either 2,22dE or 3D2,22,25dE, raising the possibility that it could occur at an early step in the biosynthetic pathway. In this respect, labelled 22R-hydroxycholesterol was efficiently converted into E and 20E, whereas 22S-hydroxycholesterol was not transformed into ecdysteroids, because of its unsuitable configuration at C22. Finally, the conversion of 25-hydroxycholesterol into E and 20E was greatly enhanced after thermal treatment of prothalli which induces the release of previously stored ecdysteroids. Thus, P. vulgare prothalli and calli appear to be particularly suitable models for the study of ecdysteroid biosynthesis and its regulation in plants.