Binding of antigen-bearing fluorescent liposomes to the murine myeloma tumor MOPC 315.

Small unilamellar lipid vesicles bearing the DNP-hapten on their surfaces and containing the water-soluble fluorescent dye carboxyfluorescein were formed by sonication. These vesicles were incubated with cells from the murine myeloma tumor MOPC 315, which secrete and also bear on the cell surface an immunoglobulin with affinity for the nitrophenyl hapten. At 0 degrees C the cells bound an average of several thousand vesicles at saturation. This binding was specific for the nitrophenyl hapten on the vesicle since it was abolished by an excess of soluble nitrophenyl derivative, by omission of the hapten from the vesicle, or by substitution for MOPC 315 of a tumor lacking receptors for the nitrophenyl hapten. Specific binding of vesicles was greater when cells were incubated at 37 degrees C. The study suggests that ligand-bearing vesicles can be a useful marker for cell surface immunoglobulin. However, in spite of the ability to "target" vesicles to cell surface determinants, binding did not result in increased delivery of vesicle contents to the cytoplasm.     

Influence of subunit-specific antibodies on the activity of the F0 complex of the ATP synthase of Escherichia coli. II. Effects of subunit c-specific polyclonal antibodies.

After incubation of F1-stripped everted membrane vesicles with antibodies against subunit c of the ATP synthase of Escherichia coli the proton translocation through the open F0 channel was blocked. Rebinding of F1 to those vesicles is affected by the antibody concentration used. In general, the use of F(ab')2 or Fab fragments prepared from anti-c antibodies gave similar results. However, using Fab fragments a higher amount of antigenic binding sites was necessary to block the F0 complex completely, whereas extremely low amounts of Fab fragments were necessary to inhibit the binding of F1. This can be explained by an antigenic determinant of subunit c, which is only accessible to the smaller Fab fragments with a molecular mass of approximately 50,000. Incubation of F1-containing everted membranes with anti-c antibodies showed that the binding of the antibodies resulted in a displacement of F1, while simultaneously the proton translocation through F0 has been blocked. Such a displacement can only be observed after incubation with IgG molecules or F(ab')2 fragments. Fab fragments were not able to displace the F1 part, indicating that the ability of antibodies and F(ab')2 fragments to produce cross-links is responsible for the loss of F1 from the membranes.     

Effects of alcohol-induced lipid interdigitation on proton permeability in L-alpha-dipalmitoylphosphatidylcholine vesicles.

6-Carboxyfluorescein was employed to examine the effect of alcohol-induced lipid interdigitation on proton permeability in L-alpha-dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles. Proton permeability was measured by monitoring the decrease of 6-carboxyfluorescein fluorescence after a pH gradient from 3.5 (outside the vesicle) to 8.0 (inside the vesicle) was established. At 20 degrees C and below 1.2 M ethanol, the fluorescence decrease is best described by a single exponential function. Above 1.2 M ethanol, the intensity decrease is better described by a two-exponential decay law. Using the fitted rate constants and the vesicle radii determined from light-scattering measurements, the proton permeability coefficient, P, in DPPC vesicles was calculated as a function of ethanol concentration. At 20 degrees C, P increases monotonically with increasing ethanol content up to 1.0 M, followed by an abrupt increase at 1.2 M. The vesicle size also exhibits a sudden increase at around 1.2 M ethanol, which has been shown to result from vesicle aggregation rather than vesicle fusion. The abrupt increases in P and in vesicle size occur at the concentration region close to the critical ethanol concentration for the formation of the fully interdigitated gel state of DPPC. At 14 degrees C, the abrupt change in P shifts to 1.9-2.0 M ethanol, completely in accordance with the ethanol-temperature phase diagram of interdigitated DPPC. Effects of methanol and benzyl alcohol on lipid interdigitation have also been examined. At 20 degrees C, DPPC large unilamellar vesicles exhibit a dramatic change in P at 3 M methanol and at 40 mM benzyl alcohol. These concentrations come close to the critical methanol and benzyl alcohol concentrations for the formation of fully interdigitated DPPC structures determined previously by others. It can be concluded that proton permeability increases dramatically as DPPC is transformed from the noninterdigitated gel to the fully interdigitated gel state by high concentrations of alcohol. This marked increase in proton permeability can be attributed to the combined effect of the changes in membrane thickness and surface charge density, due to the ethanol-induced lipid interdigitation. The possible effects of the increased proton permeability caused by ingested ethanol on gastric mucosal membranes are discussed.     

Cytochemical study of liposome and lipid vesicle phagocytosis

Electron microscopy cytochemistry has been used to study the cytoplasmic location of liposomes and lipid vesicles following specific antibody-dependent phagocytosis. The vesicle compositions were 94–99 mol% ‘fluid’ lipid (egg phosphatidylcholine or dimyristoylphosphatidylcholine at 37°C or ‘solid’ lipid (dipalmitoylphosphatidylcholine at 37°C). In some cases, 4 mol% phosphatidylserine was included in the vesicle membrane so as to vary the surface charge density. These vesicles undergo specific antibody-dependent phagocytosis by RAW264 macrophages when the lipid membranes contain 1–2 mol% dinitrophenyl lipid hapten in the presence of rabbit anti-dinitrophenyl IgG antibody. Internalized lipid vesicles can be visualized with the electron microscope when ferritin is trapped in the internal aqueous compartments prior to internalization. The lipid vesicles were demonstrated to be internal to the macrophage plasma membranes by selectively staining the plasma membranes with Ruthenium red. The cytoplasmic location of vesicles and liposomes was studied by electron microscopic staining for activities of the following enzymes: (1) acid phosphatase; (2) inorganic trimetaphosphatase; (3) adenosine triphosphatase; and (4) glucose-6-phosphatase. The first two enzymatic activities were found in association with ferritin-containing vesicles after antibody-dependent phagocytosis, showing the formation of vesicle-containing phagolysosomes. Adenosine triphosphatase and glucose-6-phosphatase were primary not associated with the vesicles, suggesting a minimal association of vesicles with plasma membrane, Golgi, endoplasmic reticulum and perinuclear cisternae. Phagosome-lysosome fusion did not appear to depend on the type of target lipid vesicle or liposome, on the ‘fluidity’ of the target membrane, or the presence of phosphatidylserine in the target membrane.     

A membrane-bound fluorescent probe to detect phospholipid vesicle-cell fusion

Summary Pyrenesulfonylphosphatidylethanolamine has been incorporated into sonicated phospholipid vesicles to provide a fluorescent signal from a membrane-bound probe whose spectrum is sensitive to the local concentration of dye molecules. When vesicle material was taken up by viable mouse splenocytes, the disappearance of the pyrene excimer fluorescence emission peak that accompanied dilution of the vesicle membrane lipid could be quantitated. One can thus measure, by a simple and rapid procedure, a new parameter which is related to the extent of vesicle-cell fusion and which is independent of the transfer of aqueous vesicle contents to the cell cytoplasm.Abbreviations used 6-CF 6-carboxyfluorescein - HBSS Hanks Balanced Salt Solution - DMPC dimyristoylphosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - EYL egg yolk lecithin - PE phosphatidylethanolamine - PEG polyethylene glycol - PLV phospholipid vesicle - PSPE pyrenesulfonylphosphatidylethanolamine     

Interaction of antibodies with liposomes bearing fluorescent haptens

Kinetic and equilibrium aspects of the recognition of antigenic model membranes by antibodies have been studied. Monoclonal anti-fluorescein IgG and its monovalent Fab fragment were allowed to interact with a fluorescein-lipid hapten that was incorporated into phospholipid vesicles. The binding was assayed in the nanomolar hapten concentration range by monitoring the quenching of hapten fluorescence by antibody. The rate and strength of the binding depended on the lipid composition of the vesicles; cholesterol enhanced both. The biphasic binding kinetics observed at high antibody concentrations for some compositions, plus additional spectroscopic evidence, led us to hypothesize that the hapten existed in a composition-dependent equilibrium between at least two conformations: (1) extended away from the membrane surface, available for binding, and (2) sequestered at or in the surface, unavailable for binding. The rate and strength of IgG binding were always greater than those of Fab, indicating bivalent binding by the IgG. This binding was intra-vesicular, since no agglutination of the vesicles was detected.     

Fusion of coated vesicles with lysosomes: measurement with a fluorescence assay

A fluorescence assay was developed to measure the rate of fusion of highly purified clathrin-coated vesicles isolated from bovine brain with purified lysosomes isolated from bovine kidney. Coated vesicles and stripped vesicles, prepared by removal of clathrin from coated vesicles with dilute alkaline buffer, were labeled with the nonfluorescent dye 6-carboxydiacetylfluorescein. Fusion of the vesicles with lysosomes resulted in mixing of the vesicle contents and exposure of 6-carboxydiacetylfluorescein to lysosomal esterases, which hydrolyze the probe's acetate groups to give the fluorescent 6-carboxyfluorescein. Fusion was therefore measured by recording the increase in fluorescence obtained upon mixing the vesicles with lysosomes. The results of the experiments indicated that the clathrin coat of coated vesicles inhibited the fusion of the vesicle membrane with that of the lysosome. In addition, fusion appears to require free Ca2+ and does not require vesicle-surface protein.     

Effects of lipid headgroup and packing stress on poly(ethylene glycol)-induced phospholipid vesicle aggregation and fusion.

The effect of lipid headgroup and curvature-related acyl packing stress on PEG-induced phospholipid vesicle aggregation and fusion were studied by measuring vesicle and aggregate sizes using the quasi-elastic light scattering and fluorescence energy transfer techniques. The effect of the lipid headgroup was monitored by varying the relative phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents in the vesicles, and the influence of hydrocarbon chain packing stress was controlled either by the relative amount of PE and PC content in the vesicles, or by the degree of unsaturation of the acyl chains of a series of PEs, e.g., dilinoleoylphosphatidylethanolamine (dilin-PE), lysophosphatidylethanolamine (lyso-PE), and transacylated egg phosphatidylethanolamine (TPE). The PEG threshold for aggregation depends only weakly on the headgroup composition of vesicles. However, in addition to the lipid headgroup, the curvature stress of the monolayer that forms the vesicle walls plays a very important role in fusion. Highly stressed vesicles, i.e., vesicles containing PE with highly unsaturated chains, need less PEG to induce fusion. This finding applies to the fusion of both small unilamellar vesicles and large unilamellar vesicles. The effect of electrostatic charge on vesicle aggregation and fusion were studied by changing the pH of the vesicle suspension media. At pH 9, when PE headgroups are weakly charged, increasing electrostatic repulsion between headgroups on the same bilayer surface reduces curvature stress, whereas increasing electrostatic repulsion between apposing bilayer headgroups hinders intervesicle approach, both of which inhibit aggregation and fusion, as expected.     

Preparation and characterization of long chain amino acid and peptide vesicle membranes

Four amino acid dicarboxylic amphiphiles which contain cysteine or homocysteine were synthesized. Each forms synthetic bilayer membranes upon hydration. Extensive sonication above the lipid phase transition temperature, 61 to 82 degrees C, produced 1000 A diameter vesicles. Treatment of the vesicles with water-soluble carbodiimides during and after sonication induced oligopeptide formation at the vesicle surface with retention of vesicle size and shape. Size exclusion chromatography indicates the products are predominantly di- to decapeptides. The permeability characteristics of the amino acid and peptide vesicles to [3H]glucose and 6-carboxyfluorescein are reported. The amino acid vesicles are among the least permeable nonpolymerized bilayer vesicles described in the literature to date. Formation of the peptide vesicles increases the membrane permeability, whereas in other polymerizable lipid vesicles the permeability decreases upon polymerization. The amino acid vesicles can be immobilized on Sephadex beads by reaction with carbodiimide. The impermeability, biodegradability, and ease of immobilization make this class of vesicles attractive materials for the encapsulation of reagents.     

Valency of CD3 binding and internalization of the CD3 cell-surface complex control T cell responses to second signals: distinction between effects on protein kinase C, cytoplasmic free calcium, and proliferation

We have studied the relationship of valency of CD3 stimulation and modulation of the CD3 receptor complex with biochemical and proliferative responses of T cells. Anti-CD3 Fab, as well as F(ab')2 and whole antibody caused rapid modulation of the CD3 antigen, whereas anti-CD3 conjugated to Sepharose did not. In the absence of monocytes, T cells stimulated with anti-CD3 Fab, F(ab')2, or F(ab')2-Sepharose showed differences in their ability to respond to second signals given by PMA, IL 1, IL 2, or antibodies to Tp67 and Tp44. None of the anti-CD3 signals alone caused resting T cells to produce IL 2, and only the Sepharose-bound anti-CD3 F(ab')2 caused T cells to express high levels of functional IL 2 receptors. Anti-CD3 F(ab')2-Sepharose-stimulated T cells produced IL 2 and proliferated in response to each of the second signals. Because anti-CD3-Sepharose did not cause modulation of the CD3 antigen, the ability of the Sepharose-bound antibody to induce T cells to express IL 2 receptors and to respond to individual second signals may be related to lack of modulation rather than valency of binding. Anti-CD3 Fab-stimulated T cells responded to PMA but required combinations of other second signals. T cells stimulated with unmodified anti-CD3 antibody or F(ab')2 fragments responded to PMA but did not respond to any other second signals alone or in combination. Stimulations that resulted in modulation (i.e., anti-CD3 whole antibody, anti-CD3 F(ab')2, or anti-CD3 Fab fragments) caused an increase in cytoplasmic calcium levels in resting T cells but blocked proliferation of T cells in response to mitogenic lectins or CD2 stimulation. Anti-CD3 F(ab')2 on Sepharose, however, did not block T cell proliferation. Whole bivalent anti-CD3 antibody or F(ab')2 fragments, but not monovalent Fab fragments, caused a rapid translation of protein kinase C activity from cytosol to membrane in the Jurkat T cell line. Because all of these modulate the receptor, these data indicate that the functional difference between monovalent and bivalent binding to CD3 is related to antibody valency and not to antigenic modulation. The use of Fab anti-CD3 stimulation that requires combinations of second signals for proliferation allowed an analysis of the functional relationships between IL 1, anti-Tp67, and anti-Tp44.     

Interaction of wheat alpha-thionin with large unilamellar vesicles.

The interaction of the wheat antibacterial peptide alpha-thionin with large unilamellar vesicles has been investigated by means of fluorescence spectroscopy. Binding of the peptide to the vesicles is followed by the release of vesicle contents, vesicle aggregation, and lipid mixing. Vesicle fusion, i.e., mixing of the aqueous contents, was not observed. Peptide binding is governed by electrostatic interactions and shows no cooperativity. The amphipatic nature of wheat alpha-thionin seems to destabilize the membrane bilayer and trigger the aggregation of the vesicles and lipid mixing. The presence of distearoylphosphatidylethanolamine-poly(ethylene glycol 2000) (PEG-PE) within the membrane provides a steric barrier that inhibits vesicle aggregation and lipid mixing but does not prevent leakage. Vesicle leakage through discrete membrane channels is unlikely, because the release of encapsulated large fluorescent dextrans is very similar to that of 8-aminonaphthalene-1,3,6,trisulfonic acid (ANTS). A minimum number of 700 peptide molecules must bind to each vesicle to produce complete leakage, which suggests a mechanism in which the overall destabilization of the membrane is due to the formation of transient pores rather than discrete channels.     

Modulation of membrane fusion by membrane fluidity: temperature dependence of divalent cation induced fusion of phosphatidylserine vesicles

We have investigated the temperature dependence of the fusion of phospholipid vesicles composed of pure bovine brain phosphatidylserine (PS) induced by Ca2+ or Mg2+. Aggregation of the vesicles was monitored by 90 degrees light-scattering measurements, fusion by the terbium/dipicolinic acid assay for mixing of internal aqueous volumes, and release of vesicle contents by carboxyfluorescein fluorescence. Membrane fluidity was determined by diphenylhexatriene fluorescence polarization measurements. Small unilamellar vesicles (SUV, diameter 250 A) or large unilamellar vesicles (LUV, diameter 1000 A) were used, and the measurements were done in 0.1 M NaCl at pH 7.4. The following results were obtained: (1) At temperatures (0-5 degrees C) below the phase transition temperature (Tc) of the lipid, LUV (PS) show very little fusion in the presence of Ca2+, although vesicle aggregation is rapid and extensive. With increasing temperature, the initial rate of fusion increases dramatically. Leakage of contents at the higher temperatures remains limited initially, but subsequently complete release occurs as a result of collapse of the internal aqueous space of the fusion products. (2) SUV (PS) are still in the fluid state down to 0 degree C, due to the effect of bilayer curvature, and fuse rapidly in the entire temperature range from 0 to 35 degrees C in the presence of Ca2+. The initial rate of leakage is low relative to the rate of fusion. At higher temperatures (15 degrees C and above), subsequent collapse of the vesicles' internal space causes complete release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric analysis of sialyl Lewis A antigen on human cancer cells by using F(ab')2μ fragments prepared from a mouse IgM monoclonal antibody

F(ab')2 fragments, herein designated as F(ab')2μ fragments, were prepared from a mouse IgM monoclonal antibody specific to sialyl Lewis A antigen. The fragments were applied to flow cytometry to analyze the antigen on human cancer cells. The binding of the fragments to the antigen-positive cells was stronger than that of the original IgM. The non-specific binding of the IgM antibody to the antigen-negative cells was much decreased by using the F(ab')2μ fragments. These results indicate that the F(ab')2μ fragments are more suitable than the original IgM monoclonal antibody in flow cytometric analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mitogenic effect on human lymphocytes of insolubilized anti-immunoglobulins. I. Specificity of the stimulating agent

The mitogenic response of peripheral blood lymphocytes to various anti-immunoglobulin reagents has been studied by measuring incorporation of a radioactive thymidine into macromolecules. Coupling of anti-F(ab')2 or anti-light chain antibodies to Sepharose beads leads to a 5-fold increase in their mitogenic capacity with 50-fold less antibodies per culture. Pepsin-digested F(ab')2 fragments had a mitogenic capacity similar to intact antibody molecules. Anti-F(ab')2 antibodies purified by immunoabsorbent columns were found to be more effective as mitogen than unpurified antibody fractions. Antibodies to kappa- or lambda-light chains were found to be mitogenic, whereas antibodies specific to various heavy chain classes failed to induce a significant response. Isolated light chains were much more effective in inhibiting the reaction than isolated mu-chains. It is concluded that insolubilized anti-light chain antibodies are mitogenic to human peripheral blood lymphocytes.     

Fusion,Leakage and Surface Hydrophobicity of Vesicles Containing Phosphoinositides: Influence of Steric and Electrostatic Effects

Calcium and lanthanum ion-induced fusion of lipid vesicles containing phosphatidylinositol (PI), phosphatidylinositol-4-monophosphate (PIP), phosphatidylinositol-4,5-bisphosphate (PIP2) or phosphatidylinositol-3,4,5-trisphosphate (PIP3) and its associated membrane properties, e.g., surface dielectric constant and vesicle leakage, were studied by fluorescence methods. The presence of poly-phosphorylated phosphoinositides (PPI) in lipid vesicles enhanced fusion, depending on the PPI phosphorylation level and the PPI concentration, as determined by the lipid mixing assay. This correlation held even at physiologically relevant small concentrations of PPI in vesicle membranes. However, the presence of nonphosphorylated PI inhibited fusion due to the steric effect of the inositol ring. The cation threshold concentration for the lipid mixing of vesicles made of mixtures of phosphatidylserine (PS) with PI increased with increasing PI contents. For all vesicle systems studied, a decrease in vesicle surface dielectric constant and an increase in vesicle leakage accompanied fusion. The presence of the nonphosphorylated inositol ring in PI did not interfere with the changes in the surface dielectric constant caused by fusogenic cations. Therefore, we deduce that the reduction of the surface dielectric constant is a necessary condition for membrane fusion to occur but it does not correlate with membrane fusion when interacting membranes are blocked for close approach as by the nonphosphorylated inositol ring.     

Inhibition of immune opsonin-independent phagocytosis by antibody to a pulmonary macrophage cell surface antigen

Unlike other hamster phagocytes, hamster pulmonary macrophages (PM) avidly ingest albumin-coated latex particles in the absence of serum. They also possess a highly specific cell surface antigen. To evaluate the relationship between these two characteristics, PM were incubated with mouse monoclonal antibody directed against the PM antigen. After unbound antibody was removed, the amount of bound antibody and the phagocytic capability of PM were measured by flow cytometry and fluorescence microscopy. Maximum antibody binding produced a 25% inhibition of ingestion. Particle attachment was not affected. This effect was antigen specific, since neither a nonspecific mouse myeloma protein of the same subclass nor a mouse antibody that bound to another hamster surface antigen had any effect on binding or ingestion. If antigen-specific F(ab')2 fragments were introduced both before and during the period of phagocytosis, the inhibition of particle ingestion approached 100%. Particle binding increased at low F(ab')2 concentrations but declined at higher concentrations. Because calcium may play a role in the ingestion process, the effect of antibody on 45Ca uptake was evaluated. It was observed that antigen-specific F(ab')2 fragments stimulated 45Ca uptake, whereas control antibodies did not. These results suggest that the antigen reacting with our anti-hamster PM monoclonal antibody is involved in immune opsonin-independent phagocytosis and that calcium participates in this phagocytic process.     

Immunoglobulin-positive mononuclear cells in human peripheral blood: detection by mixed anti-globulin and direct anti-globulin-rosetting reactions.

Ig-bearing mononuclear cells were identified in Ficoll-Hypaque preparations of human peripheral blood by using mixed anti-globulin (MAG) and direct anti-globulin rosettes; indicator cells consisted of sheep erythrocytes coated with human F(ab')2 or anti-F(ab')2 antibody, respectively. Of the cell population isolated from 10 normal subjects, a mean of 68% was lymphocytes. However, fewer than 50% of the cells with detectable surface Ig were lymphocytes. On viable cell preparations using chromic chloride-treated sheep erythrocytes (CrCl3SRBC) coated with anti-F(ab')2 antibody, a mean of 20.1% of the lymphocytes formed rosettes, i.e., were B. Up to 6% of peripheral blood lymphocytes formed mixed Ig-rosettes and E-rosettes. On viable lymphocytes using F(ab')2-coated CrCl3SRBC, MAG rosettes were insensitive in detection of B lymphocytes. Formaldehyde treatment of lymphocytes increased the number of B cells detectable to 25.5% of the lymphocyte population. Study of T-enriched and B-enriched populations showed that the observed increase in B cell reactivity was real and not due to MAG-rosetting T cells. A one-stage procedure for T and B lymphocyte separation is described.     

Lymphoma-vesicle interactions: vesicle adsorption, membrane fragmentation, and intermembrane protein transfer

Sonicated dimyristoylphosphatidylcholine vesicles interact with cultured murine lymphoma (BL/VL3) to generate complexes of vesicle and cell membrane components. Cell-free supernatants harvested after cell-vesicle incubations contain three distinct lipid species that can be separated by density gradient centrifugation. Analysis of protein and lipid composition and assays for cell and vesicle lumen contents reveal that the densest of the three lipid species comprises sealed plasma membrane fragments complexed with vesicles, while the least dense species is indistinguishable from pure phospholipid vesicles. The third, intermediate density species consists of topologically intact vesicles with associated plasma membrane proteins but without detectable cell lipids or cytoplasmic components. The membrane fragmentation and cell-to-vesicle protein transfer observed during lymphoma-vesicle incubations are examined as functions of cell and vesicle concentrations and incubation time.     

Purification of antibody and antibody-fragment from E. coli homogenate using 6,9-diamino-2-ethoxyacridine lactate as precipitation agent

To obtain a more efficient purification process for antibody fragments from an Escherichia coli homogenate, the precipitant, Ethodin (6,9-diamino-2-ethoxyacridine lactate) was introduced to the homogenate. By adding the precipitant a drastic reduction of host cell protein was obtained. The majority of the proteins were recovered in a precipitate with the cell debris, while the antibody or antibody-fragment was recovered in the clarified supernatant. In addition, DNA was also efficiently precipitated when using Ethodin as a precipitation agent. The improved purity of the clarified extract obtained by using the precipitant allows for the use of smaller chromatography columns and may reduce the number of chromatographic steps required in the recovery process. The effect of Ethodin concentration, pH, temperature, and conductivity were investigated. The investigation was performed on two different antibody-fragments, e.g., F(ab')(2) molecules and a full-length antibody produced in E. coli. The two F(ab')(2) proteins were F(ab')(2)A and F(ab')(2)B, which have a similar molecular mass (100 kDa) but different isoelectric points (pIs), i.e., 8.9 and 7.5, respectively. The full-length antibody, Ab (the full IgG form of F(ab')(2)B) has a pI of 7.8 and molecular mass of 150 kDa. The investigation showed that the highest purification factors were obtained at neutral pH, low conductivity, and Ethodin concentrations of 0.6%.     

Foreword: Translocation of proteins across membranes: systems,conservation and evolutionary origin

The interactions of mouse thymocytes with unilamellar phospholipid vesicles comprised of dimyristyl lecithin (DML), dipalmitoyl lecithin (DPL), dioleoyl lecithin (DOL), and egg yolk lecithin (EYL) were examined in vitro.

In cells treated with [³H]DML or [³H]DPL vesicles, electron microscope (EM) autoradiographic analysis showed most of the radioactive lipids to be confined to the cell surface. Transmission EM studies showed the presence of intact vesicles (DPL) and collapsed or ruptured vesicle fragments (DML) adsorbed to the surfaces of treated cells. In cells treated with DPL vesicles containing a watersoluble dye (6-carboxyfluorescein; 6-CF), most of the fluorescent vesicles were localized at the periphery of the treated cells. Furthermore, substantial fractions of the cell-associated DPL and DML could be released by a mild trypsinization without damaging the cells. These results suggest that the uptake of DML and DPL is primarily due to vesicle-cell adsorption. Such an adsorption process appears to be enhanced at or below the thermotropic-phase transition temperature of the vesicle lipid. Under certain conditions these adherent vesicles also formed patches or caps on the cell surface.

In cells treated with DOL or EYL vesicles, transmission EM and EM autoradiography showed relatively little exogenous vesicle lipid located at the cell surface. Thymocytes incubated (37°C) with [¹⁴C] EYL vesicles containing a trapped marker, [³H]inulin, incor porated both isotopes at identical rates. In separate experiments it was found that this marker was located inside the treated cells. Thymocytes treated with DOL vesicles containing 6-CF exhibited a uniform and diffuse distribution of dye in the internal volume of the cells. Little cell-associated EYL or DOL could be released by trypsinization. Evidence against endocytosis of intact vesicles as a major pathway of vesicle uptake is also presented. These observations, coupled with the demonstration of vesicle-cell lipid exchange as a minor component of vesicle uptake suggest that incorporation of EYL and DOL vesicles by thymocytes is primarily by vesicle-cell fusion.