Rheological properties of concentrated skim milk: importance of soluble minerals in the changes in viscosity during storage.

Properties of condensed milks prior to spray drying dictate to a large extent the functionality of the resulting milk powder. Rheological properties of concentrated skim milk, with total solids content of 45% but different mineral content, were studied as a function of shear rate and storage time at 50 degrees C. These milks are proposed as a model to study the effects of minerals on rheology and age gellation of condensed milk prior to drying. During storage of the concentrated milk, the apparent viscosity, particularly after 4 h, increased markedly at all shear rates studied. The yield stress also increased steeply after 4 h of storage at 50 degrees C. The changes in apparent viscosity of concentrated milk stored for up to 4 h were largely reversible under high shear, but irreversible in samples stored for longer time. The appearance of yield stress suggested the presence of reversible flocculation arising from weak attraction between casein micelles, with a transition from reversible to irreversible aggregation during storage. Particle size analysis confirmed irreversible aggregation and fusion of casein micelles during storage. Gradual reduction of mineral content of concentrated milks resulted in a marked decrease in the apparent viscosity and casein micelle aggregation during storage, while addition of minerals to milk had the opposite effect. The results demonstrated that the soluble mineral content is very important in controlling the storage-induced changes in the rheology of concentrated milks.

Protein-induced fusion can be modulated by target membrane lipids through a structural switch at the level of the fusion peptide.

Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or Golgi membranes is strongly inhibited by minor amounts of (lyso)lipids when present in the target membrane but not when inserted into the viral or Golgi membrane itself. To investigate the underlying mechanism, we employ a membrane-anchored peptide system and show that fusion is similarly regulated by these lipids when inserted into the target but not when present in the peptide-containing membrane. Peptide-induced fusion is regulated by a reversible switch of secondary structure from a fusion-permissive alpha-helix to a nonfusogenic beta-sheet. The "on/off" activation of this switch is governed by minor amounts of (lyso)-phospholipids in targets, causing a drop in alpha-helix and a dramatic increase in beta-sheet contents. Concomitantly, fusion is inhibited, due to impaired peptide insertion into the target membrane. Our observations in biological fusion systems together with the model studies suggest that distinct lipids in target membranes provide a means for regulating membrane fusion by causing a reversible secondary structure switch of the fusion peptides.

Lipid headgroup spacing and peptide penetration, but not peptide oligomerization, modulate peptide-induced fusion.

In this study, the mechanism by which an amphipathic negatively charged peptide consisting of 11 amino acids (WAE) induces fusion of liposomal phosphatidylcholine membranes is investigated. WAE-induced fusion, which only occurs when the peptide is covalently attached to the bilayer, shows a highly remarkable dependence on naturally occurring phosphatidylcholine species. The initial rate of fusion increased in the order 1-palmitoyl 2-arachidonoyl PC (PAPC) > 1-palmitoyl 2-oleoyl PC (POPC) > 1-stearoyl 2-oleoyl PC (SOPC) > dioleoyl PC (DOPC) > egg yolk PC. Interestingly, the susceptibility of the various PC species toward WAE-induced fusion matched a similar order of increase in intrinsic lipid headgroup spacing of the target membrane. The degree of spacing, in turn, was found to be related to the extent by which the fluorescence quantum yield of the Trp residue increased, which occurred upon the interaction of WAE with target membranes. Therefore, these results demonstrate an enhanced ability for WAE to engage in hydrophobic interactions when headgroup spacing increases. Thus, this latter parameter most likely regulates the degree of penetration of WAE into the target membrane. Apart from penetrating, WAE oligomerizes at the site of fusion as revealed by monitoring the self-quenching of the fluorescently derivatized lipid anchor to which WAE is attached. Clustering appears specifically related to the process of membrane fusion and not membrane aggregation. This is indicated by the fact that fusion and clustering, but not aggregation, display the same strict temperature dependence. However, evidence is presented indicating that clustering is an accompanying event rather than a prerequisite for fusion. The notion that various biologically relevant fusion phenomena are accompanied by protein clustering and the specific PC-species-dependent regulation of membrane fusion emphasize the biological significance of the peptide in serving as a model for investigating mechanisms of protein-induced fusion.

Impaired redistribution of aminophospholipids with distinctive cell shape change during Ca2+-induced activation of platelets from a patient with Scott syndrome.

We have investigated phospholipid redistribution, membrane vesicle shedding, shape change, and granule release following A23187 activation of platelets from a patient with Scott syndrome, characterized by impaired transmembrane migration of phosphatidylserine (PS) accompanied by haemorrhagic complications, and two of her children. Electron spin resonance spectroscopy measurement of phospholipids redistribution showed that the internalization of PS was unaffected by the disorder but, after activation, PS exposure was significantly reduced in platelets from the homozygous-type patient. Vesicle shedding was also reduced in these platelets. However, the slow redistribution of phosphatidylcholine was similar to that observed in normal platelets. When treated with calpeptin, platelets from the homozygous-type patient, unlike normal or heterozygous Scott syndrome platelets, showed a smoothly rounded shape without filopods after activation. Following A23187 activation of normal platelets, filopod formation was consecutive to the re-exposition of aminophospholipids on the outer leaflet of the plasma membrane, and the existence of a floppase (outward aminoPLs translocase) has been suggested. In homozygous Scott syndrome platelets the deficiency in PS re-exposition, the absence of filopod formation, and low vesicle shedding are correlated with each other, and argue in favour of a disruption of the proposed floppase activity.

Involvement of ATP-dependent Pseudomonas exotoxin translocation from a late recycling compartment in lymphocyte intoxication procedure.

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal 125I-labeled PE was transported after 2 h at 37 degrees C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20 degrees C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect 125I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when 125I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired 125I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.

Membrane fusion induced by 11-mer anionic and cationic peptides: a structure-function study.

We recently demonstrated that an amphipathic net-negatively charged peptide consisting of 11 amino acids (WAE 11) strongly promotes fusion of large unilamellar liposomes (LUV) when anchored to a liposomal membrane [Pecheur, E. I., Hoekstra, D., Sainte-Marie, J., Maurin, L., Bienvenue, A., and Philippot, J. R. (1997) Biochemistry 36, 3773-3781]. To elucidate a potential relationship between peptide structure and its fusogenic properties and to test the hypothesis that specific structural motifs are a prerequisite for WAE-induced fusion, three 11-mer WAE-peptide analogues (WAK, WAEPro, and WAS) were synthesized and investigated for their structure and fusion activity. Structural analysis of the synthetic peptides by infrared attenuated total reflection spectroscopy reveals a distinct propensity of each peptide toward a helical structure after their anchorage to a liposomal surface, emphasizing the importance of anchorage on conveying a secondary structure, thereby conferring fusogenicity to these peptides. However, whereas WAE and WAK peptides displayed an essentially nonleaky fusion process, WAS- and WAEPro-induced fusion was accompanied by substantial leakage. It appears that peptide helicity as such is not a sufficient condition to convey optimal fusion properties to these 11-mer peptides. Studies of changes in the intrinsic Trp fluorescence and iodide quenching experiments were carried out and revealed the absence of migration of the Trp residue of WAS and WAEPro to a hydrophobic environment, upon their interaction with the target membranes. These results do not support the penetration of both peptides as their mode of membrane interaction and destabilization but rather suggest their folding along the vesicle surface, posing them as surface-seeking helixes. This is in striking contrast to the behavior observed for WAE and WAK, for which at least partial penetration of the Trp residue was demonstrated. These results indicate that subtle differences in the primary sequence of a fusogenic peptide could induce dramatic changes in the way the peptide interacts with a bilayer, culminating in equally drastic changes in their functional properties. The data also reveal a certain degree of sequence specificity in WAE-induced fusion.

Membrane anchorage brings about fusogenic properties in a short synthetic peptide.

The fusogenic properties of an amphipathic net-negative peptide (wae 11), consisting of 11 amino acid residues, were studied. We demonstrate that, whereas the free peptide displays no significant fusion activity, membrane fusion is strongly promoted when the peptide is anchored to a liposomal membrane. The fusion activity of the peptide appears to be independent of pH, and membrane merging is an essentially nonleaky process. Thus, the extents of lipid mixing and contents mixing were virtually indistinguishable. Vesicle aggregation is a prerequisite for fusion. For this process to take place, the target membranes required a positive charge which was provided by incorporating lysine-coupled phosphatidylethanolamine (PElys). The coupled peptide, present in one population, could thus cause vesicle aggregation via nonspecific electrostatic interaction with PElys. However, the free peptide failed to induce aggregation of PElys vesicles, suggesting that the spatial orientation of the coupled peptide codetermined its ability to bring about vesicle aggregation and fusion. With the monitoring of changes in the intrinsic Trp fluorescence, in conjunction with KI-quenching studies, it would appear that hydrophobic interactions facilitate the fusion event, possibly involving (partial) peptide penetration. Such a penetration may be needed to trigger formation of a transient, nonbilayer structure. Since lysophosphatidylcholine inhibited while monoolein strongly stimulated peptide-induced fusion, our data indicate that wae 11-induced fusion proceeds according to a model consistent with the stalk-pore hypothesis for membrane fusion.

Transferrin receptor functions as a signal-transduction molecule for its own recycling via increases in the internal Ca2+ concentration.

Transferrin binding to its receptor modulates transferrin receptor (Tf-R) recycling rates in several cells [Klausner, R. D., Van Renswoude, J., Ashwell, G., Kempf, C., Schechter, A., Dean, A. & Bridges, K. R. (1983a) J. Biol. Chem. 258, 4715-4724; Gironès, N. & Davis, R. J. (1989) Biochem. J. 264, 35-46; Sainte-Marie, J., Vidal, M., Bette-Bobillo, P., Philippot, J. R. & Bienvenüe, A. (1991) Eur. J. Biochem. 201, 295-302]. To delineate the mechanism of this regulation, we hypothesized that the binding of the ligand to its receptor could lead to activation of several second-messenger pathways, which may redundantly stimulate recycling of the receptor. The effects of different regulators of Ca2+ flux or concentrations were investigated on the Tf-R-recycling pathway; these studies were carried out in two cell types. Perhexiline, a calcium antagonist, slowed receptor recycling in comparison with the control by more than 80% in L2C cells and by 60% in Jurkat cells (B and T lymphoblasts, respectively) but did not affect their internalization rate. Perhexiline thus trapped considerable amounts of Tf-R in the internal compartment. Ca2+ chelators, such as EGTA or 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid, and a Ca2+-channel inhibitor (Ni2+) decreased drastically the recycling rate of Tf-R. Tf-R recycling was shown to be slowed by a calmodulin antagonist. Conversely, artificial elevation of free internal Ca2+ in L2C cells, using lectin, accelerated the recycling rate. These results suggest that the intracellular Ca2+ concentration plays an important role in the outward flow of transferrin receptors. Consequently, we examined the role of transferrin in internal free Ca2+ regulation. The addition of transferrin or anti-(Tf-R) Ig specifically elicited a rise in [Ca2+], as demonstrated by inefficacy of apotransferrin or irrelevant antibodies. These results suggest that Ca2+ is a regulator of Tf-R recycling and that Tf-R seems to function as a signal-transduction molecule (perhaps in conjunction with other membrane proteins) rather than merely as an endocytic receptor.

A rapid redistribution of transferrin receptors to the cell surface of L2C lymphocytes upon fixation of holoform transferrin.

The internalization and recycling kinetics of transferrin receptors in leukemic lymphocytes (L2C) differed in the absence or presence of ligand. At 37 degrees C in the absence of ligand, transferrin receptors were mainly distributed internaly. We demonstrated, using a sepharose-bead-Tf complex, the rapid recycling of unoccupied internal transferrin receptors was correlated with ligand binding to surface receptors. The recycling amplitude was related to the occupancy of iron ligated transferrin to plasma membrane surface receptors. Contrary to the results obtained with other cells, redistribution of Tf receptors was not triggered by binding of other ligands to their receptors.

Phosphatidylserine exposure on the platelet plasma membrane during A23187-induced activation is independent of cytoskeleton reorganization.

Modifications in cytoskeleton organization (monitored by scanning electron microscopy study of platelet shape) and cytoskeleton proteolysis were investigated for their role in phosphatidylserine exposure (measured with spin-labeled analogues of phospholipids) during A23187-induced activation of human platelets. Resting platelets treated with combinations of calpeptin and cytoskeleton-disrupting agents (nocodazole or cytochalasin D) remained discoid, and there was no dense granule release, cytoskeleton proteolysis or vesicle shedding. Spin-labeled phosphatidylserine was fully and rapidly redistributed (t1/2 approximately 5 min) from the outer to the inner leaflet of the plasma membrane through ATP-dependent aminophospholipid translocase activity. In contrast, spin-labeled phosphatidylcholine was only partially and slowly redistributed (less than 20% within 60 min) to the inner leaflet. Filopod formation, vesicle shedding, and calpain-mediated proteolysis were inhibited during activation of platelets treated with calpeptin and cytoskeleton-disrupting agents. Moreover, regardless of whether platelets were treated or not, spin-labeled phosphatidylserine was rapidly (t1/2 < 1 min) and massively (50%) exposed on the outer leaflet of the plasma membrane, while the slow and slight spin-labeled phosphatidylcholine influx did not counterbalance spin-labeled phosphatidylserine outflux. These results demonstrated that phosphatidylserine exposure was not connected to the following activation-related processes: cytoskeleton modifications (actin and tubulin polymerization, submembrane skeleton proteolysis), inhibition of aminophospholipid translocase, and filopod formation. Moreover, the redistribution kinetics of spin-labeled phospholipids during activation strongly suggested the involvement of an aminophospholipid exposure mechanism that differs from a scrambling phenomenon.

Transverse redistribution of phospholipids during human platelet activation: evidence for a vectorial outflux specific to aminophospholipids.

The redistribution kinetics of phospholipids during human platelet activation by calcium ionophore were investigated to determine the specificity of the observed phospholipid outflux [Bassé et al. (1993) Biochemistry 32, 2337]. (1) Two double-labeling experiments were performed with a combination of equal amounts of spin- and fluorescently-labeled phosphatidylserine and phosphatidylcholine. During A23187-induced activation, 50% of the internal phosphatidylserine analogs were rapidly (t1/2 < 1 min) reexposed on the platelet surface while no reciprocal influx of the external phosphatidylcholine analogs was observed. (2) Treatment with chlorpromazine allowed the internalization of 20% of external spin-labeled sphingomyelin or spin-labeled phosphatidylcholine, without either inducing platelet activation or interfering with aminophospholipid translocase activity or with A23187-induced activation (dense granule secretion and vesicle shedding). During A23187-induced activation, none of the previously internalized choline head phospholipids were exposed externally, while spin-labeled phosphatidylserine outward movements were similar irrespective of whether platelets were pretreated or not pretreated with chlorpromazine. Our results demonstrated that during strong platelet activation (1) the PL excess in the internal leaflet, due to the probe addition, is not responsible for their outflux; (2) the rapid aminophospholipid outflux is definitely a vectorial outflux not counterbalanced by a rapid reciprocal influx of choline head phospholipids (i.e., not scrambling); and (3) the vectorial outflux is specific for aminophospholipids since previously internalized sphingomyelin and phosphatidylcholine did not move outward. This suggests that the specific vectorial outflux of aminophospholipids could be catalyzed by a "reverse aminophospholipid translocase" activity.

A new cationic liposome encapsulating genetic material. A potential delivery system for polynucleotides.

The endeavour to enhance gene therapy has led to increased research on the development of simple, efficient and safe delivery systems. This study deals with the use of an artificial cationic lipid on the encapsulation of genetic material in liposomes. The addition of a biologically degradable cationic phospholipid, dipalmitoyl-L-alpha-phosphatidylethanolamine covalently coupled to L-lysine, in a standard liposome formulation allowed us to obtain vesicles with high entrapment of various polynucleotides. Polynucleotide degradation by nucleases is markedly prevented by these liposomes. The preparations were stable in both culture medium and human plasma. This latter finding is consistent with the weak binding of plasma proteins on the liposome surface. The efficiency of this new delivery system was demonstrated in antiviral assays. Finally, these liposomes displayed a relatively low cellular toxicity. All these findings indicate that these cationic vesicles are very suitable for genetic material vehiculation.

Design of a short membrane-destabilizing peptide covalently bound to liposomes.

We characterized the physical and biological properties of a 14-residue amphipathic sequence called SFP (for short fusogenic peptide). At acidic pH, this short synthetic peptide interacts with various phospholipidic monolayers. These interactions were correlated with a pH-dependent conformational transition of SFP resulting in a hydrophobic alpha-helical structure. The hemolysis assay showed a pH-dependent weak membrane destabilizing activity of SFP. However, membrane anchoring of SFP through a covalently bound myristic acid enhanced by 1000-fold its membrane-destabilizing power. Moreover, SFP covalently bound to fluorescent-labeled liposomes induced a pH-dependent mixing of both membranes. SFP, a small synthetic peptide, is thus able to mimick many aspects of viral protein-induced membrane fusion: conformational change, membrane destabilization, membrane anchoring and finally pH-dependent lipid mixing.

Loss of phospholipid asymmetry in human platelet plasma membrane after 1-12 days of storage. An ESR study.

We used paramagnetic analogs of endogenous phospholipids to study modification of phospholipid distribution in platelet plasma membranes during aging. Asymmetrical distributions and translocation kinetics were very different for spin-labeled phosphatidylserine and spin-labeled phosphatidylcholine in fresh platelet plasma membranes. In freshly prepared platelets and up to day 7, spin-labeled phosphatidylserine very rapidly penetrated to the inner leaflet of the platelet plasma membrane. However, spin-labeled phosphatidylcholine was mainly retained on the external leaflet. From day 7 to day 9, the two translocation kinetics became identical with symmetrical distribution of both spin-labeled phospholipids at equilibrium. Inhibition of translocase activity and modification of membrane stability accounted for these transformations. The rapid re-exposition of spin-labeled phosphatidylserine after stimulation by the calcium ionophore A23187, measured in fresh platelet concentrates, persisted up to day 9 but disappeared between day 10 and day 12. From day 7 to day 9, a strong cytoskeleton proteolysis and marked decrease in intracellular ATP were observed. Moreover, complete suppression of beta-N-acetyl glucosaminidase secretion and vesicle formation after A23187 stimulation of aged platelets indicated that platelets could no longer be activated beyond day 9. Taken together, these results showed that during in vitro aging there are metabolic and membrane modifications in platelet similar to those described for platelet activation. In addition, all of the observed events occurred simultaneously between day 7 and day 9. These results highlight the importance of maintaining plasma membrane asymmetry to increase the hemostatic effectiveness of transfused platelet concentrates.

Correlation between inhibition of cytoskeleton proteolysis and anti-vesiculation effect of calpeptin during A23187-induced activation of human platelets: are vesicles shed by filopod fragmentation?

Platelets were incubated in the presence of calpeptin to inhibit calpain-mediated cytoskeleton proteolysis during further activation by Ca2+ ionophore A23187. The appearance of filamin and myosin subfragments (93 kDa and 135 kDa, respectively) was inhibited by low calpeptin doses (1 microgram/ml). Higher doses (10-20 micrograms/ml) were required to completely inhibit talin and filamin degradation. Vesiculation strongly depended on cytoskeleton proteolysis and was reduced by 60% when platelets were preincubated with 10 micrograms/ml calpeptin. Activated platelets bore longer and more filopods when pretreated with calpeptin. Filopods were straight and regular when high calpeptin doses were used, whereas they were shorter and broader with bloated surfaces when calpeptin was omitted. Some bloated areas were also found in straight filopods. These results suggest that the cytoskeleton proteolysis, and more specifically filamin proteolysis, induced bloating of filopod surfaces, thus facilitating fragmentation of filopod into vesicles.

Translocation of spin-labeled phospholipids through plasma membrane during thrombin- and ionophore A23187-induced platelet activation.

After incorporation of spin-labeled phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine analogues in the outer leaflet of the plasma membrane in resting platelets, more than 90% amino-head analogues accumulated within 30 min in the inner leaflet by aminophospholipid translocase activity, while choline analogues mostly remained on the outer leaflet. Platelets were then activated by thrombin or Ca2+ ionophore A23187. No outward movement of internally located spin-labeled aminophospholipids was observed during thrombin-induced activation, whereas the influx of externally located probes increased slightly. During A23187-mediated activation, similar slightly increased influx was observed, while 40-50% of the initially internally located aminophospholipids could then be extracted from the outer leaflet. This sudden exposure on the outer face was dependent on an increase in intracellular Ca2+ and achieved in less than 2 min at 37 degrees C. Inhibition of translocase activity by N-ethylmaleimide did not induce any aminophospholipid outflux. When probes were incorporated on the outer face of the plasma membrane in resting platelets, they were still fully accessible from the extracellular medium after A23187-induced activation. Moreover, they were distributed between the vesicles and remnant platelets in proportion to the external membrane phospholipidic content in each structure. This suggested that no scrambling of plasma membrane leaflets occurred during the vesicle blebbing. Moreover, the spin-labeled aminophospholipids exposure rate and amplitude were unchanged when vesicle formation was inhibited by the calpain inhibitor calpeptin. These results indicate that loss of asymmetry thus inducing generation of a catalytic surface is not the consequence of vesicle formation. Conversely, we propose that vesicle shedding is an effect of PL transverse redistribution and calpain-mediated proteolysis during activation.

Phospholipid transverse mobility modifications in plasma membranes of activated platelets: an ESR study.

Spin labeled phospholipid analogs were used to directly study changes in aminophospholipid translocase activity in activated platelets. In thrombin-activated platelets, the translocase activity was slightly stimulated, whereas no vesicle formation or proteolysis of cytoskeletal protein occurred. Ca2+ ionophore A23187-mediated activation produced vesiculation and proteolysis. Additionally, the translocase activity was completely inhibited, probably due to a sharp rise the intracellular Ca2+ concentration, as shown when platelets were activated in the presence of various A23187 and Ca2+ concentrations and by the recovery of the translocase activity when Ca2+ was complexed with EGTA. No translocase activity was found in vesicles. Whereas vesiculation and translocase inhibition can occur independently of proteolysis, this later accentuated the shedding phenomenon.

Interferon production of L929 and HeLa cells enhanced by polyriboinosinic acid-polyribocytidylic acid pH-sensitive liposomes.

The double-stranded RNA polyinosinic acid-polycytidylic acid (PolyIC) is an inducer of interferons alpha and beta (IFN) genes. With L929 and HeLa cells IFN pretreatment (priming) improves the IFN induction by PolyIC by several orders of magnitude. In the absence of the priming we demonstrate that PolyIC encapsulated into pH-sensitive liposomes (and not into pH-insensitive liposomes) enables L929 cells to secrete IFN efficiently and a low toxicity is observed; on primed cells pH-sensitive liposomes containing PolyIC trigger a high toxicity. With HeLa cells, the absence of the priming PolyIC encapsulated into pH-sensitive liposomes induces weak doses of IFN whereas free PolyIC was ineffective. Our experiments established that a pH drop (from 8 to 5.5) provoked a lipid mixing between pH-sensitive liposomes and cell membranes, likely by a fusion mechanism. Entrapment into pH-sensitive liposomes enhances the effect of PolyIC by several orders of magnitude, which might improve its therapeutic ability as an antitumor or anti-HIV agent.

Selective translocation of the A chain of diphtheria toxin across the membrane of purified endosomes.

Translocation is a necessary and rate-limiting step for diphtheria toxin (DT) cytotoxicity. We have reconstituted DT translocation in a cell-free system using endosomes purified from lymphocytes and have demonstrated this using two different probe/cell systems, which provided identical results: 125I-DT/human CEM cells and 125I-transferrin-DT/mouse BW cells. The cell-free DT translocation process was found to be dependent on the presence of the pH gradient endosome (pH 5.3)/cytosol (pH 7). Among the pH equilibrating agents, nigericin (5 microM) was found to be the most effective, inhibiting DT translocation by 88%. An optimum pH value of 7 on the cytosolic side of the membrane (pH gradient approximately 1.7) was determined. ATP per se is not required for DT translocation. 125I-DT translocation was 3-fold more active from late than from early endosomes, probably because of their slightly more acidic pH. Only the A chain of the toxin was found to escape from either 125I-DT/CEM or 125I-transferrin-DT/BW endosomes. Translocation of control endosome labels (125I-transferrin and 125I-horseradish peroxidase) was never observed. We also show that DT receptors present on resistant (mouse) cells block the translocation of the toxin and are responsible for the resistance of these cells to DT.

Targeting of poly(rI)-poly(rC) by fusogenic (F protein) immunoliposomes.

The aim of this study is to investigate the intracellular delivery of polynucleotides by fusogenic immunoliposomes. We have studied the internalization of poly(rI)-poly(rC) (polyIC) by liposomes into murine L929 cells. The liposomes were prepared by incorporating Sendaï virus fusogenic F protein into the lipid bilayer and targeted by a monoclonal antibody (mAb) bound to the liposomes via protein A (Staphylococcus aureus). The immunoliposomes ensured a sufficient yield of polyIC internalization, which was estimated by its ability to induce antiviral activity. In the absence of RNase treatment free and encapsulated polyIC had the same inducing effect, but in the presence of nuclease only the encapsulated polyIC, and not free polyIC, maintained its antiviral effect. The fusion process making possible the internalization of polyIC was confirmed by the fact that the polyIC effect was mainly inhibited by an anti-F protein mAb which inhibited erythrocyte hemolysis by the virus.