Elicitins secreted by phytopathogenic Phytophthoraspp. are proteinaceous elicitors of plant defense mechanisms and were demonstrated to load, carry, and transfer sterols between membranes. The link between elicitor and sterol-loading properties was assessed with the use of site-directed mutagenesis of the 47 and 87 cryptogein tyrosine residues, postulated to be involved in sterol binding. Mutated cryptogeins were tested for their ability to load sterols, bind to plasma membrane putative receptors, and trigger biological responses. For each mutated elicitin, the chemical characterization of the corresponding complexes with stigmasterol (1:1 stoichiometry) demonstrated their full functionality. However, these proteins were strongly altered in their sterol-loading efficiency, specific binding to high-affinity sites, and activities on tobacco cells. Ligand replacement experiments strongly suggest that the formation of a sterol-elicitin complex is a requisite step before elicitins fasten to specific binding sites. This was confirmed with the use of two sterol-preloaded elicitins. Both more rapidly displaced labeled cryptogein from its specific binding sites than the unloaded proteins. Moreover, the binding kinetics of elicitins are related to their biological effects, which constitutes the first evidence that binding sites could be the biological receptors. The first event involved in elicitin-mediated cell responses is proposed to be the protein loading with a sterol molecule.

Cosmetic products need to have a proven efficacy combined with a comprehensive toxicological assessment. Before the current Cosmetic regulation N°1223/2009, the 7th Amendment to the European Cosmetics Directive has banned animal testing for cosmetic products and for cosmetic ingredients in 2004 and 2009, respectively. An increasing number of alternatives to animal testing has been developed and validated for safety and efficacy testing of cosmetic products and cosmetic ingredients. For example, 2D cell culture models derived from human skin can be used to evaluate anti-inflammatory properties, or to predict skin sensitization potential; 3D human skin equivalent models are used to evaluate skin irritation potential; and excised human skin is used as the gold standard for the evaluation of dermal absorption. The aim of this manuscript is to give an overview of the main in vitro and ex vivo alternative models used in the safety testing of cosmetic products with a focus on regulatory requirements, genotoxicity potential, skin sensitization potential, skin and eye irritation, endocrine properties, and dermal absorption. Advantages and limitations of each model in safety testing of cosmetic products are discussed and novel technologies capable of addressing these limitations are presented.

Angiotensin II (Ang II) and adrenocorticotropic hormone stimulate aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex through induction of the expression of the steroidogenic acute regulatory (StAR) protein, which promotes intramitochondrial cholesterol transfer. To understand the mechanism of this induction of the StAR protein, we have examined the effect of Ang II and forskolin, a mimicker of adrenocorticotropic hormone action, on two transcription factors known to modulate StARgene expression in opposite ways, DAX-1 and SF-1, in bovine adrenal glomerulosa cells in primary culture. Ang II markedly inhibited DAX-1 protein expression in a time- and concentration-dependent manner (to 38.7 ± 12.9% of controls at 3 nm after 6 h, p < 0.01), an effect that required de novo protein synthesis and ERK2/1 activation. This effect was associated with a concomitant decrease in DAX-1 mRNA and an increase in mitochondrial StAR protein levels. Similarly, forskolin dramatically repressed DAX-1 protein and mRNA expression (to 19.6 ± 1.8 and 50.3 ± 4.7% of controls, respectively,p < 0.01). Neither Ang II nor forskolin affected DAX-1 protein and mRNA stability. The aldosterone response to Ang II was markedly reduced (to 59 ± 4% of controls,p < 0.01) in transiently transfected cells overexpressing DAX-1. Whereas Ang II was without effect on SF-1 expression, forskolin significantly increased SF-1 protein and mRNA levels in a cycloheximide-sensitive manner (to 167.4 ± 16.6 and 173.1 ± 25.1% of controls after 6 h, respectively,p < 0.01). These results demonstrate that the balance between repressor and inducer function of DAX-1 and SF-1 are of critical importance in the regulation of adrenal aldosterone biosynthesis.