Cytotoxic activity of a tumor protease-activated pore-forming toxin.

Equinatoxin II is a pore forming toxin produced by the sea anemone Actinia equina. It is able to kill very unspecifically most cell types by the membrane-perturbing action of an amphiphilic alpha-helix located at its N-terminal. A normally active N-terminal mutant, containing one single cys in the amphiphilic alpha-helix, becomes totally inactive when it is bound to avidin via a biotinylated linker. By choosing, as a linker, a peptide containing a tumor protease cleavage site, we were able to construct an enzymatically activable conjugate which should be selective for tumor cells. The introduced cleavage site was designed in order to be digested by both cathepsin B and matrix metalloproteases (MMPs). We confirmed that this conjugate could be activated in vitro by cathepsin B and MMPs. After having measured the enzymatic activity of fibrosarcoma and breast carcinoma cells, we analyzed the cytotoxic effect of the conjugate on the same lines and on human red blood cells (HRBC) as controls. We found that the conjugate was activated, at least in part, by the tumor cell lines used, whereas it was inactive on HRBC. That the activation process was dependent on the enzymatic action of cathepsin B and MMPs, was indicated by three lines of evidence: (1) binding occurred normally on all type of cells including HRBC which however were insensitive being devoid of enzymes; (2) the cytotoxic effect correlated with the amount of cathepsin B activity expressed by the cells; (3) conjugate activation was reduced by specific inhibitors of cathepsin B and MMPs. These results demonstrate the possibility of tumor cell killing by a pore-forming toxin conjugate specifically activated by tumor proteases.

Structure-function studies of tryptophan mutants of equinatoxin II, a sea anemone pore-forming protein.

Equinatoxin II (EqtII) is a eukaryotic cytolytic toxin that avidly creates pores in natural and model lipid membranes. It contains five tryptophan residues in three different regions of the molecule. In order to study its interaction with the lipid membranes, three tryptophan mutants, EqtII Trp(45), EqtII Trp(116/117) and EqtII Trp(149), were prepared in an Escherichia coli expression system [here, the tryptophan mutants are classified according to the position of the remaining tryptophan residue(s) in each mutated protein]. They all possess a single intrinsic fluorescent centre. All mutants were less haemolytically active than the wild-type, although the mechanism of erythrocyte damage was the same. EqtII Trp(116/117) resembles the wild-type in terms of its secondary structure content, as determined from Fourier-transform infrared (FTIR) spectra and its fluorescent properties. Tryptophans at these two positions are buried within the hydrophobic interior of the protein, and are transferred to the lipid phase during the interaction with the lipid membrane. The secondary structure of the other two mutants, EqtII Trp(45) and EqtII Trp(149), was altered to a certain extent. EqtII Trp(149) was the most dissimilar from the wild-type, displaying a higher content of random-coil structure. It also retained the lowest number of nitrogen-bound protons after exchange with (2)H(2)O, which might indicate a reduced compactness of the molecule. Tryptophans in EqtII Trp(45) and EqtII Trp(149) were more exposed to water, and also remained as such in the membrane-bound form.

Characterization of hemolytic activity of 3-alkylpyridinium polymers from the marine sponge Reniera sarai.

Polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai act as potent anticholinesterase agents; in addition they show moderate hemolytic and cytotoxic activities. The hemolytic activity of poly-APS is due to their detergent-like structure and behavior in aqueous solutions. In this work, the hemolytic activity of poly-APS is analyzed and compared to that of structurally-related monomeric cationic surfactants. The influence of different divalent cations and lipids on poly-APS induced hemolysis is discussed. The dimensions of lesions caused by poly-APS in erythrocyte membranes are determined by the use of osmotic protectants. Finally, the possible role of poly-APS in their natural environment is proposed.

A comparison of the symptoms associated with early and late onset alcohol dependence.

This study was designed to determine whether the prevalences of the DSM-III alcohol abuse/dependence symptoms in 87 early and 73 late onset male alcoholics differ from one another. The authors administered a 19-item alcohol abuse/dependence symptom checklist with items based on the DSM-III criteria. Nine of the 19 symptoms were reported significantly more often in the early than in the late onset alcoholics. Antisocial behaviors were reported to have been particularly frequent in the early onset group.

A comparative outcome study of frequent, moderate, occasional, and nonattenders of Alcoholics Anonymous.

The purpose of this project was to identify the outcomes associated with frequent, moderate, occasional, and nonparticipation in Alcoholics Anonymous by male alcohol dependents during the first month after treatment. Informants reported nonparticipants consumed far more alcohol during a 48 week followup than moderate or occasional participants. Moderate and occasional participants were rated as abstinent more often than nonparticipants. Nonparticipants were also reported jailed more often than participants. All other consumption and quality of life comparisons between the groups were nonsignificant. Occasional and moderate AA attendance appear to be associated with better outcomes than nonattendance, but frequent participation was not associated with additional improvement.

A 48-week natural history follow-up of alcoholics who do and do not engage in limited drinking after treatment.

The research on the controversial Alcoholics Anonymous tenet that limited drinking rapidly leads alcoholics to inebriety is inconclusive. We conducted 48-week follow-ups on 51 posttreatment alcohol dependents who had reportedly engaged in limited drinking and 51 paired controls who apparently had not. According to the informants, the limited drinkers consumed 16 times as much alcohol and were 4 times as likely to regress to unacceptable drinking as controls. They were also more often rehospitalized and attended fewer Alcoholics Anonymous meetings than the controls. They were, however, usually (62%) categorized as abstinent or moderate drinkers when assessed during the follow-up period. The groups did not differ in risk of jailing, detoxification, or job loss, nor did limited drinkers ordinarily regress quickly to inebriety. The outcomes of our limited drinkers were inferior to those of controls but much less negative than those Wilson's Alcoholics Anonymous maintains.