HAMLET forms annular oligomers when deposited with phoshpolipid monolayers

Recently, the anticancer activity of human α-lactalbumin made lethal to tumor cells (HAMLET) has been linked to its increased membrane affinity in vitro, at neutral pH, and ability to cause leakage relative to the inactive native bovine α-lactalbumin (BLA) protein. In this study, atomic force microscopy resolved membrane distortions and annular oligomers (AOs) produced by HAMLET when deposited at neutral pH on mica together with a negatively charged lipid monolayer. BLA, BAMLET (HAMLET's bovine counterpart) and membrane-binding Peptide C, corresponding to BLA residues 75–100, also form AO-like structures under these conditions but at higher subphase concentrations than HAMLET. The N-terminal Peptide A, which binds to membranes at acidic but not at neutral pH, did not form AOs. This suggests a correlation between the capacity of the proteins/peptides to integrate into the membrane at neutralpH—as observed by liposome content leakage and circular dichroism experiments—and the formation of AOs, albeit at higher concentrations. Formation of AOs, which might be important to HAMLET's tumor toxic action, appears related to the increased tendency of the protein to populate intermediately folded states compared to the native protein, the formation of which is promoted by, but not uniquely dependent on, the oleic acid molecules associated with HAMLET.publishedVersio

In thrombin stimulated human platelets Citalopram, Promethazine, Risperidone, and Ziprasidone, but not Diazepam, may exert their pharmacological effects also through intercalation in membrane phospholipids in a receptor-independent manner

Intercalation of drugs in the platelet membrane affects phospholipid-requiring enzymatic processes according to the drugs’ intercalation capability. We investigated effects of Promethazine, Citalopram, Ziprasidone, Risperidone, and Diazepam on phospholipase A2 (PLA2) and polyphosphoinositide (PPI) metabolism in thrombin-stimulated human platelets. We also examined effects of the drugs on monolayers of glycerophospholipids using the Langmuir technique. Diazepam did not influence PLA2 activity, had no effects on PPI cycle, and caused no change in mean molecular area of phospholipid monolayers. The remaining psychotropic drugs affected these parameters in different ways and levels of potency suggesting that they act by being intercalated between the molecules of adjacent membrane phospholipids, thus causing changes in substrate availability for phospholipid-hydrolyzing enzymes (PLA2 and Phospholipase C). We show that several psychotropic drugs can also have other cellular effects than receptor antagonism. These effects may be implicated in the psychotropic effects of the drugs and/or their side effects

HAMLET forms annular oligomers when deposited with phoshpolipid monolayers

Recently, the anticancer activity of human α-lactalbumin made lethal to tumor cells (HAMLET) has been linked to its increased membrane affinity in vitro, at neutral pH, and ability to cause leakage relative to the inactive native bovine α-lactalbumin (BLA) protein. In this study, atomic force microscopy resolved membrane distortions and annular oligomers (AOs) produced by HAMLET when deposited at neutral pH on mica together with a negatively charged lipid monolayer. BLA, BAMLET (HAMLET's bovine counterpart) and membrane-binding Peptide C, corresponding to BLA residues 75–100, also form AO-like structures under these conditions but at higher subphase concentrations than HAMLET. The N-terminal Peptide A, which binds to membranes at acidic but not at neutral pH, did not form AOs. This suggests a correlation between the capacity of the proteins/peptides to integrate into the membrane at neutralpH—as observed by liposome content leakage and circular dichroism experiments—and the formation of AOs, albeit at higher concentrations. Formation of AOs, which might be important to HAMLET's tumor toxic action, appears related to the increased tendency of the protein to populate intermediately folded states compared to the native protein, the formation of which is promoted by, but not uniquely dependent on, the oleic acid molecules associated with HAMLET

Potentiation by adrenaline of thrombin-induced elevation of pHi is not essential for synergistic activation of human platelets

AbstractGel-filtrated human platelets were stimulated with thrombin in the absence and presence of adrenaline. Adrenaline markedly enhanced the thrombin-induced increase in cytoplasmic pH (pHi) in BCECF-loaded platelets. This rise in pHi was strongly inhibited by the Na+/H+ exchange blocker EIPA. The potentiation by adrenaline of thrombin-induced PLC activation measured as [32P]PA formation and final platelet responses was, however, not blocked by EIPA, even at low concentrations of thrombin. These results indicate that the enhancement by adrenaline of thrombin-induced cytoplasmic alkalinization may be a secondary effect which is not essential for the potentiation by adrenaline of platelet activation by thrombin