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Positively charged liposomes have been shown to inhibit the proliferation of lymphocytes induced by various polyclonal activators. We demonstrated that this inhibition is essentially restricted to early phases of activation. B cell proliferation, induction of suppressor cells, and cytotoxic activities are all profoundly inhibited, whereas T4+ cells response to mitogenic stimulation is only moderately affected. The results are discussed in terms of membrane perturbations potentially induced by liposome-lymphocyte interactions.

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The effect of toxic-shock-syndrome-toxin-1 (TSST-1) on coagulation and platelet aggregation was studied in blood samples from human healthy volunteers. TSST-1 at 5 micrograms/ml does not modify the extrinsic or intrinsic coagulation pathways. However, thrombin clotting time (TCT) was significantly increased in the presence of TSST-1. Platelet aggregation was not directly affected by TSST-1 but the toxin strongly inhibited platelet aggregation induced either by epinephrine, adenosine diphosphate (ADP) or platelet activating factor (PAF), while having no effect on aggregation induced by thrombin, collagen or a calcium ionophore. The above results suggest that TSST-1 may bind to a transducer common for some aggregators or that it may induce some non-specific modification of platelet membrane.

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The efficiency of positively-charged liposomes as immunomodulators was investigated in vitro using a lymphoproliferative response assay on cultured human lymphocytes challenged either with different polyclonal activators or with actin, a cytoskeletal protein of poor immunogenicity. Our results indicate that these liposomes suppress the [3H]thymidine incorporation in lymphocyte cultures and inhibit the mitogen-induced proliferation without, however, exhibiting significant cytotoxicity.

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The Ca2+ sensitivity of liver gelactin-induced actin gelation was reinvestigated by low-shear viscosity using the falling-ball technique. By this technique, we demonstrate that the gelatin of actin by gelactin can be influenced by the presence of calcium ions depending on the concentrations of both proteins, actin and gelactin. At low concentrations of gelactin, the gelatin of actin exhibits a bell-shaped dependency on free calcium ion concentration, being stimulated between pCa 8 and 6 and inhibited at pCa below 5.5, while at high gelactin concentrations the calcium sensitivity of actin gelation is apparently abolished. Although the sensitivity observed in the physiological range of calcium concentrations may be of importance in vivo, the sensitivity observed at higher calcium concentrations more probably reflects the state of actin polymerization in different ionic conditions. These results confirm our previous conclusions on the peculiarity of gelactin as an F-actin cross-linker.

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Electron microscopy, ultracentrifugation, gel filtration, and isoelectric focusing were carried out with gelactin, an actin-gelling protein from rabbit liver. Gelactin is a dimeric acidic protein (isoelectric point (pI) = 5.45), with a molecular weight of 190 000, a Svedberg constant of 6.25, and a Stoke's radius and length of 7.0 and 28 nm, respectively. While different from alpha-actinin by pI and amino acid composition, gelactin belongs by its dimensions to the class of alpha-actinins.

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Actin-membrane interactions have been studied using purified liver plasma membranes and muscular filamentous actin. Despite the large quantity of endogenous actin present in membranes, exogenous muscular filamentous actin cosediments with membranes after a 30 min centrifugation at 30 000 g. The cosedimentation process is time-dependent and exhibits a complex relationship with actin concentration. The cosedimentation of actin with membranes can be partly explained by gelation as shown by low-shear viscosity and electron microscopy. The characterization of the gelation phenomenon as a function of time, actin and membrane concentrations, ionic strength, temperature and Ca2+ concentration is also presented. Gelation alone cannot however account for the overall cosedimentation data, and a more direct mode of association between actin and the membrane must be envisaged. The analogy that exists between the results obtained with liver plasma membranes and those obtained with other membrane systems suggests that a general mechanism may be involved in the interaction of actin with plasma membranes.

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An F-actin crosslinking protein, tentatively named gelactin, has been isolated from rabbit liver. This protein is a dimer of 200 000 molecular weight which promotes the lateral association of actin filaments. The effect is maximal at a molar ratio of gelactin:actin of about 1:1000. The dependency of the gelation process on various experimental factors was studied by a centrifugation assay. The results demonstrate that gelation is greatly influenced by pH, ionic strength and temperature, but is insensitive to the presence of calcium ions. We show that, in our conditions, the association of actin with gelactin does not interfere with actin-myosin interactions and does not influence the kinetics of actin polymerization. The amino acid composition of gelactin is also given. From its overall properties gelactin is shown to be different from all other actin-gelling proteins.

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G-actin incubated in presence of a liver fraction enriched in plasma membranes rapidly denatures, as evidenced by the biphasic loss of polymerizability and DNase inhibition. The inactivation is shown to result from the loss of actin-bound nucleotide induced by the rapid destruction of free nucleotides by membrane NPases. This is further supported by the observation that addition of either ATP or ADP to actin that has been exposed to membranes completely stops the denaturation process and partly restores polymerizing capacity. The biphasic aspect of inactivation is explained by the protective action of AMP and (or) adenosine formed in the course of the incubation.

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G-actin incubated in the presence of a liver fraction enriched in plasma membranes is rapidly inactivated, as indicated by the biphasic loss of polymerizability and DNase inhibition. The rates of inactivation as measured by viscosity are greatly influenced by temperature, but almost independent of membrane concentrations at least in the low range of concentrations tested (less than 250 micrograms protein/ml). The loss of DNase inhibition capacity proceeds at rates two to three times slower than the loss of polimerizability. The inactivation of actin in the presence of membranes cannot be attributed to proteolysis nor to a phosphorylation of actin by membranes. However, it is shown that in the course of the incubation, medium ATP is rapidly converted into AMP and adenosine and that the destruction of ATP is almost complete at the start of the inactivation process. A mechanism is presented relating the destruction of ATP to actin inactivation.

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Cyclic peptides from the deadly mushroom Amanita virosa has been separated by methanolic extraction and chromatography on Sephadex. Three groups of peptides have been obtained: virotoxins, amaninamide and phalloidin. Virotoxins has been separated in two fractions named virotoxins A and virotoxins B. We have studied the properties of these two fractions of F actin in vitro and on mice in vivo. Our results show that virotoxins A and B protect F actin in vitro against chaotropic ions, depolymerization by DNAse I or cytochalasin B and heat denaturation. Virotoxins A and B increase the rate of polymerization of F actin. Virotoxins A and B are toxic compounds which produce hemorrhagic necrosis of liver that have been observed in detail by electron microscopy. In general, our in vitro and in vivo results show that virotoxins exhibit the same effect as phalloidin on F actin.

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Osmium tetroxide and potassium permanganate destroy F-actin filaments in vitro, and split actin molecules into smaller peptides. It is shown by viscometry, spectrophotometry, electrophoresis and electron microscopy that the destruction of F-actin by these agents is inhibited by phalloidin. Other proteins are unprotected.

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Even at low concentration, phalloidin shows a marked protection of F-actin against the action of trypsin or pronase. G-actin is not protected at any concentration of phallodin. The kinetics of the proteolysis show that a change in the environment of tryptophan residues is preceded by disruption of the filamentous structure of F-actin.

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A method of affinity chromatography based on the trapping of actin filaments within agarose gel beads is described. This method can be used for the purification of myosin and its active proteolytic subfragments, as well as for studies on the interaction between actin and these proteins. Actin columns stabilized by phalloidin bind myosin, heavy meromyosin (HMM), and heavy meromyosin subfragment 1 (HMM-S1) specifically and reversibly. The effect of pyrophosphate and KCl on the dissociation of actomyosin, acto-HMM, or acto-HMM-S1 complex is reported. We also describe the single-step purification of myosin from a crude rabbit psoas muscle extract.

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