Giuliano D'Agnolo

Micellar gangliosides mediate the lipid insertion of cholera toxin protomer A

The topology of the interaction of cholera toxin with ganglioside and detergent micelles was studied with the technique of hydrophobic photolabelling. Cholera toxin alpha and gamma polypeptide chains appear to penetrate into the hydrophobic core of ganglioside micelles. Micelles of SDS cause the labelling also of the beta polypeptide chains, while Triton X-100 micelles have little ability to mediate the labelling of the toxin. The specific reduction of the alpha-gamma disulfide bond allows the penetration of the alpha polypeptide chain into Triton X-100 micelles, but does not affect the interaction of cholera toxin with either ganglioside or SDS micelles. Thus, ganglioside micelles appear to cause a conformational change of the native toxin, such as to induce the penetration of the alpha chain into the micelle hydrophobic core.

The role of environmental parameters on the stability of cholera toxin functional regions.

1. Introduction Native cholera toxin is composed of three unique polypeptide chains (Y, fl and 7 [l--3]. The (Y and y chains, of which there is one per molecule, are covalently linked through a single disulfide bridge, forming protomer A. The remainder of the molecule consists of five 0 chains [4], forming a stable complex designated protomer B. Thus, the toxin has a molec- ular formula w&. Both protomer A as well as poly- peptide (Y stimulate adenylate cyclase in intact cells and cell-free systems [5]. Although the mode of action of the toxin is poorly understood, it is known that protomer A, in the presence of a large excess of dithiothreitol, catalyzes the hydrolysis of NAD to ADP-ribose and nicotinamide [6,7], and the transfer of the ADP-ribosyl moiety to itself [8]. The role of the ADP-ribosylated A protomer, thus formed, in adenylate cyclase activation remains to be determined. The B protomer is devoid of any enzymatic activity but binds the toxin to its cell membrane receptor, the monosialoganglioside G,, [9-l l] and is believed to be needed for the penetration of A into the cell [3]. Exposure of the toxin to thiol reducing agents splits the molecule into two fragments, an insoluble one the (Y polypeptide chain and one left in the super- natant as a complex of the two chains /3 and y [2 1. The study of the chemical and functional properties of the two fragments is complicated by the self- association of the (Y chain after reduction. Protomer A also is unusually insoluble in aqueous media. At

Dissociation of cholera toxin functional regions after interaction with vesicles containing ganglioside GM1

immunoelectron microscopy have provided evidence that the latent period may represent the time required for a redistribution of the toxin-receptor complexes in the cell membrane [ 10,111. Whether the active functional region penetrates the membrane through channels [ 121 or a change in the conformation of the toxin [ 131, resulting from the mobility of the toxin--s1 complexes in the membrane, remains to be determined. At present, although some authors have reported an interaction of hydrophobic nature between protomer A and membranes [13,14], no direct evidence of the dissociation either of protomer A or of the (Y chain has been obtained, after binding of the toxin to biological membranes. In view of these observations, we examined the characteristic and specific binding of cholera toxin to vesicles containing GM1 [15] with respect to the possible interactions of the active (Y fragment with model membranes. The effect of binding on the reactivity of the interchain disulfide bond connecting the Q! and 7pS functional regions was also studied. This communication shows that the interaction of cholera toxin with GM1--vesicles is strong enough to induce perturbations in the lipid membrane but it does not affect the dissociation of the active (Y chain from the vesicle-bound toxin.

Common criteria among States for storage and use of dried blood spot specimens after newborn screening

Biological samples collected in biobanks are a resource with significant research potential. The Italian Joint Group CNB - CNBBSV (National Committee of Bioethics - National Committee for Biosecurity, Biotechnologies and Life Sciences) published a document reporting recommendations on storage and use of dried blood spot (DBS) and on the development of a National Network of Regional Newborn Screening Repositories for collection of residual DBS. Several ethical questions (about consent, possible use of genetic information, unanticipated possible usages for research purposes) rise from residual newborn screening specimens collections. Moreover, legal and ethical controversies are accentuated by the conflicts between the interests of sample donors, biobank holders, researchers and the public. To overcome these difficulties the identification of a few criteria for storage and research usage of DBS is crucial.

Nerve growth factor and lipid classes in sensory ganglia.

Abstract 1. 1. The effect of the nerve growth factor on the lipids of 8-day-old chickembryo sensory ganglia has been studied. Total lipids increased, and the ganglionic dry weight remained almost unchanged. 2. 2. Separation of neutral lipids showed that a rise in triglyceride content was accompanied by an increase in esterified and total cholesterol, whereas free fatty acids and a sterol-like compound diminished. 3. 3. Total phospholipids were only slightly affected, but sphingomyelin decreased significantly. A certain tendency towards lyso compounds was observed. 4. 4. The mode of action of the nerve growth factor in the light of the presented results has been discussed as an effect on enzymes concerned with neutral lipid synthesis.