Search PubMed⌕ Search

Biomedical subjects

R Blumenthal

Publications and source records attributed to R Blumenthal.

At least 127 records · Page 7Linked to original sources
PubMed1986

Polylysine induces pH-dependent fusion of acidic phospholipid vesicles: a model for polycation-induced fusion.

Polylysine induced aggregation and fusion of negatively charged small unilamellar phosphatidylcholine vesicles containing at least 10% anionic lipid. Aggregation was followed by absorbance changes and fusion was assayed both by electron microscopy and by fluorescence energy transfer between lipid probes. A method for preparing asymmetric vesicles, where the fluorescent probes were present only in the inner monolayer of the vesicle membrane, was developed. These vesicles were used to distinguish the inner and outer monolayer when measuring lipid mixing between vesicles. Since polylysine induced lipid mixing of both monolayers equally, fusion of these vesicles did occur. The extent of fusion was dependent on the charge ratio between bound polylysine and phosphatidylserine (PS) in the outer monolayer and was optimal at a ratio of about 1:1. Excess polylysine inhibited fusion. At a given concentration of polypeptide, fusion increased as the pH was lowered toward 3 with an apparent pKa near 4. Since this value is close to the pKa of the PS-carboxyl groups and far from the pKa of the lysine epsilon-amino groups, the pH dependence observed for fusion resides in the lipids rather than in the peptide. Fusion was dependent on the available lysine and not the size or molarity of the polypeptide. The data indicate that there must be sufficient sites on the vesicles and sufficient polypeptide to achieve effective aggregation. For fusion to occur after aggregation, charges on the vesicles must be neutralized either by polypeptide-PS interaction or by protonation of the PS carboxyl groups. Optimal conditions for fusion occur when charge neutralization is possible without completely covering the vesicles with polypeptide. The results are consistent with the notion that the polypeptide is necessary for fusion because of requirements for crosslinking, but limits fusion by steric inhibition.

Cations, Divalent↗
PubMed1986

pH-dependent lysis of liposomes by adenovirus.

Purified adenovirus induced a dose-dependent release of the water-soluble markers calcein and carboxyfluorescein from liposomes. Marker release was strongly dependent on pH, and at temperatures below 5 degrees C, the rate of release showed an optimum at a pH of about 6. This pH dependence parallels disruption of endocytic vesicles by adenovirus and the permeabilization that adenovirus induces on the cell surface. There did not seem to be a striking dependence on the lipid composition of the liposomes. Electron microscopy using a negative stain shows liposomes bound to adenovirus. In some cases, the liposomes were still intact, but many liposomes, which were attached to the vertices of the virus, appeared lysed. These data support the notion that adenovirus, which enters the host cell by receptor-mediated endocytosis, gains access to the cytoplasm by a subsequent pH-dependent disruption of the membrane of the endocytic vesicle.

Adenoviruses, Human↗
PubMed1986

Sizing and separation of liposomes, biological vesicles, and viruses by high-performance liquid chromatography.

The ability of an HPLC gel exclusion column (TSK G6000PW) to separate lipid vesicles, viruses, and biological vesicles according to size was tested and compared with separations on Sephacryl S1000. The columns were calibrated using vesicular Stokes radii determined by quasielastic light scattering. The vesicles separated according to size on both types of column and remained intact during elution. Viruses of known diameters and clathrin-coated vesicles were also eluted as a function of size. The TSK G6000PW column was able to separate larger particles (greater than 500 nm) than the Sephacryl S1000, and, when used in combination with the TSK G5000PW column, gave more discrete separations of smaller particles (10 to 30 nm diameter). Moreover, the HPLC columns can be run significantly faster (10-20 min vs several hours) and give more precise results than Sephacryl S1000. Therefore, HPLC using a G6000PW column alone, or in combination with a G5000PW column, provides a rapid and accurate means of sizing and selecting specifically sized biological and artificial vesicles.

Acrylic Resins↗
PubMed1986

Apocytochrome c induces pH-dependent vesicle fusion.

The ability of apocytochrome c and the heme containing respiratory chain component, cytochrome c, to induce fusion of phosphatidylcholine (PC) small unilamellar vesicles containing 0-50 mol % negatively charged lipids was examined. Both molecules mediated fusion of phosphatidylserine (PS):PC 1:1 vesicles as measured by energy transfer changes between fluorescent lipid probes in a concentration- and pH-dependent manner, although cytochrome c was less potent and interacted over a more limited pH range than the apocytochrome c. Maximal fusion occurred at pH 3, far below the pKa of the 19 lysine groups contained in the protein (pI = 10.5). A similar pH dependence was observed for vesicles containing 50 mol % cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylinositol (PI) in PC but the apparent pKa values varied somewhat. In the absence of vesicles, the secondary structure of apocytochrome c was unchanged over this pH range, but in the presence of negatively charged vesicles, the polypeptide underwent a marked conformational change from random coil to alpha-helix. By comparing the pH dependencies of fusion induced by poly-L-lysine and apocytochrome c, we concluded that the pH dependence derived from changes in the net charge on both the vesicles and apocytochrome c. Aggregation could occur under conditions where fusion was imperceptible. Fusion increased with increasing mole ratio of PS. Apocytochrome c did induce some fusion of vesicles composed only of PC with a maximum effect at pH 4. Biosynthesis of cytochrome c involves translocation of apocytochrome c from the cytosol across the outer mitochondrial membrane to the outer mitochondrial space where the heme group is attached. The ability of apocytochrome c to induce fusion of both PS-containing and PC-only vesicles may reflect characteristics of protein/membrane interaction that pertain to its biological translocation.

Animals↗
PubMed1985

Gangliosides do not move from apical to basolateral plasma membrane in cultured epithelial cells.

Both qualitative and quantitative approaches were used to ascertain whether gangliosides, incorporated into the apical plasma membrane of cultured epithelial cells from kidney of toad (A6) and dog (MDCK), were able to redistribute past the tight junctions to the basolateral membrane. The apical surfaces of confluent epithelia were exposed to rhodaminyl gangliosides and the distribution of the inserted gangliosides was assessed qualitatively by fluorescence microscopy. All of the fluorescence was confined to the apical surface for at least 1 h after the fluorescent gangliosides had become incorporated; none appeared on the basolateral surface. These observations were confirmed by incubating the cells with anti-rhodamine antibodies and 125I-labeled protein A. In order to quantitate further the ganglioside distribution, binding assays were performed using 125I-labeled cholera toxin, which binds specifically to ganglioside GM1. Exogenous GM1 added to the apical membrane was not detected on the basolateral membrane 4 h after its incorporation even though there was extensive disappearance of the inserted ganglioside, presumably through endocytosis. To directly examine the behaviour of endogenous gangliosides, the apical surface of the epithelial cells was exposed to bacterial neuraminidase, which hydrolyzes more complex gangliosides to GM1. The cells exhibited a 10-fold increase in binding of cholera toxin to their apical surface, but no increase in binding to their basolateral surface. Thus, no cellular pathways for movement from apical to basolateral plasma membrane appear to be available for implanted or endogenous gangliosides.

Animals↗
PubMed1985

Fusion of Sendai virions with phosphatidylcholine-cholesterol liposomes reflects the viral activity required for fusion with biological membranes.

Sendai virus envelopes were reconstituted after solubilization of intact virions with either Triton X-100 or octylglucoside. Envelopes obtained from Triton X-100, but not from octylglucoside solubilized virions, were hemolytic and promoted cell-cell fusion. Fluorescence dequenching studies [using N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine-bearing viral envelopes] revealed that both preparations fused with negatively charged phospholipids. Fusion with phosphatidylcholine (PC)/cholesterol (chol) liposomes was promoted only by the hemolytic viral envelopes. Fluorescence dequenching studies, using intact virions bearing octadecylrhodamine B chloride, revealed that intact virions fused with PC/chol as well as with negatively charged phospholipids. Only fusion with PC/chol liposomes was inhibited by phenylmethylsulfonyl fluoride and dithiothreitol, reagents which are known to block the viral ability to fuse with biological membranes.

Cholesterol↗
PubMed1985

The interaction of apolipoprotein A-I with small unilamellar vesicles of L-alpha-dipalmitoylphosphatidylcholine.

The interaction between apolipoprotein A-I and small unilamellar vesicles of dipalmitoylphosphatidylcholine at the lipid phase transition resulted in complete release of vesicle contents at molar ratios of lipid to protein from 4000:1 down to 50:1. This indicated the existence of two types of stable complexes: a vesicular apo-A-I complex with a maximum of two to three apo-A-Is/vesicle, and a micellar complex (disc) with a stoichiometry of about 50 phosphatidylcholines/apo-A-I (mol/mol). We characterized the complexes by density gradient centrifugation, by gel filtration, and by immunoprecipitation using an anti-apo-A-I antibody. The morphology of the discs was similar to that of previously reported discs. Apo-A-I-induced release of vesicle contents was monitored by the relief of self-quenching of vesicle-encapsulated carboxyfluorescein. Using this assay we characterized the nature of the interaction between apo-A-I and phospholipid vesicles. The formation of complexes between vesicles and apo-A-I followed a two-step process; below or above the lipid phase transition temperature (Tc), apo-A-I bound to phosphatidylcholine vesicles but caused little leakage of contents. Kinetic analysis of the interaction between apo-A-I and dipalmitoylphosphatidylcholine vesicles below Tc indicated that about 1 in 500 collisions leads to a stable apo-A-I-vesicle complex. The second step involved passage of those complexes through Tc, which resulted in a very rapid transition into discs or vesicular complexes. Vesicular complexes contain apo-A-I which was no longer capable of interacting with pure lipid. Discs, on the other hand, interacted with vesicles at their phase transition.

Apolipoprotein A-I↗
PubMed1985

The use of cobalt ions as a collisional quencher to probe surface charge and stability of fluorescently labeled bilayer vesicles.

Co2+ quenched the fluorescence of the lipid probes NBD-phosphatidylethanolamine (NBD-PE) and lissamine-rhodamine phosphatidylethanolamine (N-Rh-PE) incorporated into lipid vesicles, according to a collisional quenching mechanism in agreement with the Stern-Vollmer law. The quenching coefficient (Q) for NBD-PE, incorporated into uncharged phosphatidylcholine (PC) vesicles was 13.8 M-1. This value was equal to the quenching coefficient of water-soluble NBD-taurine in aqueous solution, indicating that Co2+ was readily accessible to the outer surface of PC vesicles. In phosphatidylserine-phosphatidylethanolamine (PS-PE) (1:1) vesicles, quenching was also proportional to Co2+ concentration but Q was 114 mM-1, some 8000-fold smaller. Using the Gouy-Chapman-Stern model we demonstrated that the surface density of Co2+ bound to lipid was linear with Co2+ concentration in the medium up to 7%. Co2+-associated phospholipid would in turn quench NBD-PE or N-Rh-PE by collisional quenching with lateral diffusion. We investigated the ability of Co2+ to permeate PS-PE (1:1) vesicles. Co2+ quenched fluorophores on the outer surface of large unilamellar vesicles, formed by reverse-phase evaporation. In small unilamellar vesicles Co2+ quenched probes on both outer and inner surfaces, indicating rapid permeation of the ions into the vesicles. Using stopped-flow rapid mixing, we measured the rate of influx of Co2+, and correcting for surface potential using the Gouy-Chapman-Stern model, we calculated a permeability coefficient of 10(-12) cm/s for Co2+ concentrations below 300 microM. Above this concentration, there was a very steep rise in the permeability coefficient, indicating that binding of Co2+ induces defects in the bilayer of these vesicles. This may be related to the ability of the vesicles to undergo membrane fusion. A method for calculating the membrane surface potential from Co2+ quenching data is presented.

Cobalt↗
PubMed1985

Rapid kinetics of Ca2+-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles. The effect of bilayer curvature on leakage.

We have employed both small unilamellar vesicles (SUV) and large unilamellar vesicles formed by the reverse phase evaporation technique (REV) to study the initial kinetics of membrane aggregation and fusion. Stopped flow measurements of the calcium-induced changes in the turbidity of SUV and REV, formed from 1:1 (mol/mol) mixtures of bovine phosphatidylserine (PS) and Escherichia coli phosphatidylethanolamine (PE), were used to follow particle aggregation. Simultaneous measurements of the fluorescence resonance energy transfer from N-(7-nitro2,1,3-benzoxadiazol-4-yl) (NBD)-PE to rhodamine (Rho)-PE incorporated into the vesicle bilayers established that 1) both initial aggregation and fusion can be described as a bimolecular process and 2) the rate-limiting step of membrane fusion is aggregation. Thus fusion takes place in the microsecond time domain. Parallel experiments, which simultaneously measured aggregation and the dequenching of encapsulated carboxyfluorescein (CF) in the presence and absence of antifluorescein antibodies in the suspension medium, established that the small unilamellar vesicles rapidly lose their contents of CF as they fuse. On the other hand, the first few cycles of fusion of the large unilamellar vesicles are nonleaky, but leakage develops within 1-2 s as the particles grow in size. Thus the results demonstrate that the SUV are poor models for the study of nonleaky fusion, while the REV must be carefully tested before unambiguous interpretation of fusion assays involving the formation of tight complexes (such as the terbium-dipicolinic assay) can be made. NBD-PE undergoes very rapid, Ca2+-promoted changes in quantum yield which can obscure the resonance energy transfer signals. Thus data from the NBD-PE/Rho-PE energy transfer pair must be carefully scrutinized for artifacts.

Animals↗
PubMed1984

pH-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstituted into phospholipid vesicles.

Purified G-protein from vesicular stomatitis virus was reconstituted into egg phosphatidylcholine vesicles by detergent dialysis of octyl glucoside. A homogeneous population of reconstituted vesicles could be obtained, provided the protein to lipid ratio was high (about 0.3 mol % protein) and the detergent removal was slow. The reconstituted vesicles were assayed for fusion activity using electron microscopy and fluorescence energy transfer. The fusion activity mediated by the viral envelope protein was dependent upon pH, temperature, and target membrane lipid composition. Incubation of reconstituted vesicles at low pH with small unilamellar vesicles containing negatively charged lipids resulted in the appearance of large cochleate structures, as shown by electron microscopy using negative stain. This process did not cause leakage of a vesicle-encapsulated aqueous marker. The rate of fusion was pH-dependent with a pK of about 4 and the apparent energy of activation for the fusion was 16 +/- 1 kcal/mol. G-protein-mediated fusion showed a large preference for target membranes which contain phosphatidylserine or phosphatidic acid. Inclusion of 36% cholesterol in any of the lipid compositions had no effect on the rate of fusion. These reconstituted vesicles provide a system to study the mechanism of pH-dependent fusion induced by a viral spike protein.

Animals↗
PubMed1984

Characteristics of cyclic nucleotide dependent regulation of cytoplasmic E2 binders in cultured endometrial and breast cells.

Estradiol binding levels have previously been shown to be increased or decreased by the addition of of cGMP or cAMP respectively to cellular homogenates prepared from human endometrial tissue or from the endometrial cell line HEC 1. Similar cyclic nucleotide dependent effects have now been demonstrated in homogenates prepared from a second endometrial cell line, HEC 50, and from two breast cancer cell lines, CG-5 and T47D. It was previously shown that ATP and Mg2+ are necessary for the generation or inactivation of E2 binding sites. The present study demonstrates that the concentration of K+ and of dithiothreitol can also affect these reactions.

Breast Neoplasms↗
PubMed1984

Liposomes as targets for granule cytolysin from cytotoxic large granular lymphocyte tumors.

Purified cytoplasmic granules from rat large granular lymphocyte tumors having natural killer activity and/or antibody-dependent cell-mediated cytotoxicity induced a rapid, dose-dependent release of the water-soluble marker carboxyfluorescein from liposomes made of phosphatidylcholine. A solubilized, partially purified cytolytic preparation termed "cytolysin" from these granules showed identical properties. Marker release induced by granules or the cytolysin was strongly dependent on the presence of Ca2+ at a concentration of 0.1 mM or higher in the medium; Ca2+ could be replaced by higher concentration of Sr2+ but not by Ba2+ or by Mg2+. These properties strikingly parallel the lytic effects that granules and granule cytolysin exert on cells. Marker release from liposomes was stopped instantaneously when an excess of EGTA was added to the medium. The remaining carboxyfluorescein inside the liposomes was present at the original internal concentration, indicating that marker release was all-or-none from individual liposomes. Liposomes comprised of lipid in the solid phase released marker more slowly than did comparable liposomes containing fluid-phase lipids. Variation of the lipid headgroup had only minor effects on the cytolysin-induced marker release. Electron microscopy of liposomes exposed to cytolysin in the presence of Ca2+ showed cylindrical structures of 15-nm diameter inserted into the membrane concomitant with the penetration of negative stain into the liposome. These properties of large granular lymphocyte granule cytolysin strongly suggest that it operates through a mechanism similar to the membrane attack of complement.

Animals↗
PubMed1984

Detection of membrane cholesterol by filipin in isolated rat liver coated vesicles is dependent upon removal of the clathrin coat.

We investigated the cholesterol content of highly purified populations of coated vesicles from rat liver by biochemical quantitation and by cytochemical electron microscopy using the polyene antibiotic filipin. Failure of this reagent to elicit its typical response for a cholesterol-containing membrane, i.e., a characteristically corrugated or rippled appearance by thin section analysis, had led to the hypothesis (Montesano, R., A. Perrelet, P. Vassalli, and L. Orci, 1979, Proc. Natl. Acad. Sci. USA., 76:6391-6395) that cholesterol is specifically excluded from the plasma membrane domains associated with coated pit regions. The present electron microscopic results showed that although the response of coated vesicle membranes to filipin was also negative, uncoated vesicles whose clathrin coats had been removed in vitro exhibited a strong filipin-positive response. Quantitated biochemically, the cholesterol-to-phospholipid ratio of the coated vesicles was found to be indistinguishable from that of control preparations of plasma membranes isolated from rat liver. Taken together, the results indicate that the filipin-negative response of coated vesicles (and probably also that of coated pits) is due not to abnormally low cholesterol content, but rather to the stabilizing influence of their enveloping clathrin coats which inhibit the characteristic structural expression of the filipin-cholesterol complexes.

Animals↗
PubMed1984

Mechanism of tetanolysin-induced membrane damage: studies with black lipid membranes.

Tetanolysin produced similar rates of leakage of K+ and hemoglobin from erythrocytes. When studied by using cholesterol-containing black lipid membranes, this hemolysin induced conductance steps with a broad frequency distribution. These findings are inconsistent with the formation of structural channels and suggest that tetanolysin acts by causing lipid perturbations.

Bacterial Toxins↗
PubMed1984

Regulation of estrogen receptor levels in endometrial cancer cells.

Recent experimental results from our laboratories revealed the following facts: Addition of GMP to homogenates or cytosol prepared from endometrial tissue or cultured endometrial adenocarcinoma cells during the assay for specific estrogen binders markedly increases specific binding levels. The effect is completed in about 15 min at 4 C (Fleming et al, 1983). Cyclic AMP has the opposite effect and in many cases lowers the number of binding sites to undetectable levels. ATP, a nucleotide that stimulates a particulate form of guanylate cyclase, Na2MoO4, a compound that can elevate cGMP levels (Fleming and Blumenthal, unpublished) and GTP, a metabolic precursor of cGMP, increase specific estradiol binding in the presence of plasma membranes and soluble factors. Cyclic AMP reduces the levels of estrogen binding when added to cell homogenates or to cytosol and counteracts the effects of cGMP, MoO4, ATP and GTP. ATP is required for the expression of cGMP and cAMP effects on estradiol binding. It is therefore likely that phosphorylations are involved in the generation and inactivation of estrogen binding sites. Divalent cation requirements for these effects also suggest participation of protein kinases in these processes. The reported effects of nucleotides and molybdate have been observed in specimens of histologically normal endometrium, in specimens of endometrial carcinoma, in two endometrial adenocarcinoma cell lines, HEC-1 and HEC-50 (Suzuki et al, 1980), and in two breast cancer cell lines, CG-5, a variant of MCF-7 obtained in Iacobelli's laboratory (Natoli et al, 1983), and in T47D) (Fleming et al, in press) Rapid changes in the levels of estrogen binding capacity observed in endometrial cells in culture can be associated with changes in cGMP/cAMP ratios shown, to vary during the cell cycle. Although it has not yet been demonstrated that cGMP-induced increases in specific estrogen binding can enhance responses to available estrogens, such possibility is of potential importance. Reduction of estrogen receptor levels in patients with cancers of estrogen sensitive tissues may inhibit tumor growth promoted by endogenous estrogen. Cho-Chung et al have recently reported that cholera toxin causes a reduction in estrogen receptor levels and arrests hormone dependent growth of DMBA-induced mammary carcinoma in rats (Cho-Chung et al, 1983). They postulated that the effect of cholera toxin is mediated by a cAMP effect on the estrogen receptor, an hypothesis supported by the observation that only tumors containing receptor responded to treatment. Conversely, cGMP-induced increases in specific estrogen binders may be useful in promoting a response of tumors to estr

Adenocarcinoma↗