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R Blumenthal

Publications and source records attributed to R Blumenthal.

At least 109 records · Page 6Linked to original sources
PubMed1990

Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy.

We have used intensified video fluorescence microscopy and digital image processing to observe and quantitate influenza virus (A/PR8/34/H1N1) fusion to human erythrocyte membranes. Viruses labeled with the lipid probe octadecylrhodamine B (R18) were seen to undergo fluorescence dequenching and eventual disappearance after exposure to pH levels known to induce virus-cell membrane fusion. Quantitative intensity measurements of single individual particles were possible. From these fluorescence data it has been possible to calculate the fraction of R18 dye molecules transferred from the virus to the cell. The redistribution of the lipid probe upon fusion at pH 5.0 had a t1/2 of 46 s, longer than expected for a free-diffusion model. The R18 loss was approximately twice as fast at pH 5.0 as at pH 5.1. No obvious delay until the start of fluorescence dequenching was observed after the pH changes, suggesting that activation processes are faster than the time resolution, 1-5 s, of the current method.

Erythrocyte Membrane↗
PubMed1990

Fusion of intra- and extracellular forms of vaccinia virus with the cell membrane.

The membrane fusion activities of the isolated single-envelope intracellular form of vaccinia virus (INV) and the double-envelope extracellular (EEV) form were studied by using a lipid-mixing assay based on the dilution of a fluorescent probe. Fluorescently labeled INV and EEV from both the IHD-J and WR strains of vaccinia virus fused with HeLa cells at neutral pH, suggesting that fusion occurs with the plasma membrane during virus entry. EEV fused more efficiently and with faster kinetics than INV: approximately 50% of bound EEV particles fused over the course of 1 h, compared with only 25% of the INV particles. Fusion of INV and EEV was strongly temperature dependent, being decreased by 50% at 34 degrees C and by 90% at 28 degrees C. A monoclonal antibody to a 14-kilodalton envelope protein of INV that has been implicated in the fusion reaction (J. F. Rodriguez, E. Paez, and M. Esteban, J. Virol. 61:395-404, 1987) completely suppressed the initial rate of fusion of INV but had no effect on the fusion activity of EEV, suggesting that vaccinia virus encodes two or more membrane fusion proteins. Finally, cells infected with the WR strain of vaccinia virus formed syncytia when briefly incubated at pH 6.4 or below, indicating that an acid-activated viral fusion protein is expressed on the cell surface. However, WR INV and EEV did not display increased fusion activity at acid pH, suggesting that the acid-dependent fusion factor is not incorporated into virions or that its activity there is masked.

Animals↗
PubMed1990

Conformational changes and fusion activity of influenza virus hemagglutinin of the H2 and H3 subtypes: effects of acid pretreatment.

Marked differences were observed between the H2 and H3 strains of influenza virus in their sensitivity to pretreatment at low pH. Whereas viral fusion and hemolysis mediated by influenza virus X:31 (H3 subtype) were inactivated by pretreatment of the virus at low pH, influenza virus A/Japan/305/57 (H2 subtype) retained those activities even after a 15-min incubation at pH 5.0 and 37 degrees C. Fusion with erythrocytes was measured by using the octadecylrhodamine-dequenching assay with both intact virions and CV-1 monkey kidney cells expressing hemagglutinin (HA) on the plasma membrane. To study the nature of the differences between the two strains, we examined the effects of low-pH treatment on the conformational change of HA by its susceptibility to protease digestion, exposure of the fusion peptide, and electron microscopy of unstained, frozen, hydrated virus. We found that the respective HA molecules from the two strains assumed different conformational states after exposure to low pH. The relationship between the conformation of HA and its fusogenic activity is discussed in the context of these experiments.

Erythrocytes↗
PubMed1990

Apical membrane vesicles of ADH-stimulated toad bladder are highly water permeable.

Antidiuretic hormone (ADH) stimulation of the toad urinary bladder causes intracellular vesicles called aggrephores to fuse with the apical plasma membrane of granular cells. Aggrephore membranes contain particle aggregates. Particle aggregates are believed to be water channels that cause large increases in the water permeability (PF) of the granular cell apical membrane. Removal of ADH causes the retrieval of particle aggregate-containing apical membrane via endocytosis and a decline in PF. We have previously shown that fluid phase markers are sequestered in these particle aggregate-containing vesicles during retrieval of the apical membrane and that these vesicles can be recovered in cell homogenates. We have now loaded these vesicles with the self-quenching fluorophore carboxyfluorescein (CF) to measure and compare their PF with that of CF-loaded resealed human erythrocyte ghosts. The membranes of these retrieved vesicles have a very high water permeability. The minimum PF of 99% of these vesicles is 4.5 X 10(-2) cm/s. This PF is comparable with that of erythrocyte ghosts (5.4 X 10(-2) cm/s) measured under identical conditions. We conclude that these vesicles are highly permeable to water, and this is consistent with their postulated function of retrieving water channels that have been inserted into the apical membrane in response to ADH.

Animals↗
PubMed1989

Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells. Measurement by dequenching of fluorescence.

Fusion between membranes of 3T3 fibroblasts expressing hemagglutinin (HA) from the Japan strain of influenza virus and human red blood cells (RBC) was measured using an assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecylrhodamine (R18). The probe was incorporated into the membrane of intact RBC at self-quenching concentrations, and the RBCs were bound to the 3T3 cells. Fusion, which allowed movement of R18 into 3T3 cell membranes, was monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, the fluorescence increased after a delay of about 30 s at 37 degrees C, and leveled off within 2 min. In control experiments where R18 RBCs bound to 3T3 cells expressing the uncleaved precursor hemagglutinin (HA0) were incubated at 37 degrees C and low pH, no fluorescence increase was observed. This indicated that the R18 dequenching occurred as a result of HA-induced fusion of plasma membranes. Fusion showed a very steep pH dependence with a threshold at pH 5.4 and a maximum at pH 5.0, similar to HA-induced fusion seen previously using cell biological techniques. The fusion rate increased and the delay for the onset of fusion decreased as the temperature was raised above 20 degrees C. Low pH activation of the fusion process at 37 degrees C could be partially arrested by raising the pH after 2-10 s, but not after 15 s, indicating that the irreversible pH-activated conformational change of HA necessary for fusion was complete within about 15 s. Analysis of the data indicates that the pH-induced membrane fusion activity of HA is a highly cooperative event.

Animals↗
PubMed1989

pH-dependent fusion of reconstituted vesicular stomatitis virus envelopes with Vero cells. Measurement by dequenching of fluorescence.

Reconstituted vesicular stomatitis virus (VSV) envelopes were formed by solubilization of the viral envelope with Triton X-100 followed by removal of detergent by direct addition of SM2 biobeads. We provide direct demonstration of fusion of reconstituted VSV with cells using fluorescent lipid and aqueous probes incorporated into the VSV virosomes during reconstitution. We show a direct comparison of the kinetics and pH profile of fusion with cells between reconstituted VSV and fluorescently labeled intact virus. With this preparation it is now possible to gain additional information about the role of cooperativity in viral protein-mediated fusion, and to permit construction of efficient vehicles for delivery of drugs and other materials into cells.

Animals↗
PubMed1989

Diminished corticosterone levels in nude mice implanted with MCF-7 or ZR-75-1 human breast tumor cells.

Corticosteroid levels were studied in the plasma of athymic mice implanted with human breast tumor cells, either from MCF-7 or ZR-75-1 cell lines. There was a highly significant decrease in plasma corticosterone levels in the mice implanted with these tumor cells. There was no significant effect on corticosterone of GW 39 colon cancer cells, LS 174T colon cancer cells, or Calu-3 lung cancer cells.

Animals↗
PubMed1989

On the use of self-quenching fluorophores in the study of membrane fusion kinetics. The effect of slow probe redistribution.

In glycoprotein-mediated pH-induced fusion of virus to animal cells, the mixing of materials between membranes or between cytoplasmic spaces occurs after the virus-cell complex has gone through a number of activation reactions. The monitoring of the fluorescence changes measured in a fusing system using self-quenching probes could reflect not only the kinetics of activation, but also the redistribution reaction of probes. For instance, time delay seen in the onset of fluorescence changes after triggering the fusion reaction (S.J. Morris, D.P. Sarkar, J.M. White and R. Blumenthal, J. Biol. Chem. (1989) 3972), could be due to rate-limiting probe redistribution kinetics. In this paper we examined in detail the effect of probe redistribution rates on fusion kinetics. Simulations were performed using a very simple model with two fusion-activation steps and an exponential probe redistribution kinetics. We conclude that if the rates of probe redistribution are faster than or equal to those of viral glycoprotein activation, the kinetics of the fusion reaction are not significantly affected.

Animals↗
PubMed1989

Initial stages of influenza hemagglutinin-induced cell fusion monitored simultaneously by two fluorescent events: cytoplasmic continuity and lipid mixing.

We have monitored the mixing of both aqueous intracellular and membrane-bound fluorescent dyes during the fusion of human red blood cells to influenza hemagglutinin-expressing fibroblasts using fluorescence spectroscopy and low light, image-enhanced video microscopy. The water-soluble fluorescent dye, N-(7-nitrobenzofurazan-4-yl)taurine, was incorporated into intact human red blood cells. The fluorescence of the dye in the intact red blood cell was partially quenched by hemoglobin. The lipid fluorophore, octadecylrhodamine, was incorporated into the membrane of the same red blood cell at self-quenching concentrations (Morris, S. J., D. P. Sarkar, J. M. White, and R. Blumenthal. 1989. J. Biol. Chem. 264: 3972-3978). Fusion, which allowed movement of the water-soluble dye from the cytoplasm of the red blood cell into the hemagglutinin-expressing fibroblasts, and movement of octadecylrhodamine from membranes of red blood cell to the plasma membrane of the fibroblasts, was observed by fluorescence microscopy as a spatial relocation of dyes, and monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, fluorescence increased after a delay of about 30 s at 37 degrees C, reaching a maximum within 3 min. The kinetics, pH profile, and temperature dependence were similar for both fluorescent events measured simultaneously, indicating that influenza hemagglutinin-induced fusion rapidly establishes bilayer continuity and exchange of cytoplasmic contents.

Animals↗
PubMed1988

Composition of octyl glucoside-phosphatidylcholine mixed micelles.

The composition of mixed micelles of egg phosphatidylcholine (PC) and octyl glucoside was studied by a novel technique based on measuring resonance energy-transfer efficiency between two fluorescent lipid probes present in trace amounts. Equations were derived for calculating the stoichiometry of the composition of mixed micelles from the energy-transfer measurements. These were applied to determining the average number of lipid molecules in the octyl glucoside-egg PC mixed micelle as a function of detergent concentration. The average number of detergent molecules in these mixed micelles was independent of lipid concentration in the range studied (0-500 microM). The dependence of mixed micelle stoichiometry on the concentration of aqueous (monomeric) octyl glucoside is consistent with the assumptions of ideal mixing of the two amphiphiles in the mixed micelles and that mixed micelles can be treated as a distinct phase.

Energy Transfer↗
PubMed1988

Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH.

Fusion of vesicular stomatitis virus (VSV) with Vero cells was measured after exposure of the virus to low pH under a variety of experimental conditions. The method of relief of fluorescence self-quenching of the probe octadecylrhodamine was used to monitor fusion. Incubation of the virus at pH 5.5 prior to binding to cells led to significant enhancement of fusion at the plasma membrane, whereas fusion via the endocytic pathway was inhibited. Fusion of pH 5.5-pretreated VSV showed a similar pH threshold for fusion as nontreated virus, and it was blocked by antibody to VSV G protein. Activation of VSV by pretreatment at low pH was only slightly dependent on temperature. In contrast, when VSV was first bound to target cells and subsequently exposed at 4 degrees C to the low pH, activation of the fusion process did not occur. The pH 5.5-mediated activation of VSV could be reversed by returning the pH to neutral in the absence of target membranes. The low pH pretreatment also led to aggregation of virus; large aggregates could be pelleted by low speed centrifugation and only the effects of the supernatant, which consist of single virions and/or microaggregates, were considered. The data were analyzed in the framework of an allosteric model according to which viral spike glycoproteins undergo a pH-dependent conformational transition to an active (fusion-competent) state. Based on that analysis we conclude that the conformational transition to the active state is rate-limiting for fusion and that the viral spike glycoproteins are fusion-competent only in their protonated form.

Allosteric Regulation↗
PubMed1988

Micelle-vesicle transition of egg phosphatidylcholine and octyl glucoside.

The dissolution and formation of egg phosphatidylcholine (PC) vesicles by the detergent octyl glucoside were examined systematically by using resonance energy transfer between fluorescent lipid probes, turbidity, and gel filtration chromatography. Resonance energy transfer was exquisitely sensitive to the intermolecular distance when the lipids were in the lamellar phase and to the transitions leading to mixed micelles. Turbidity measurements provided information about the aggregation of lipid and detergent. Several reversible discrete transitions between states of the PC-octyl glucoside system were observed by both methods during dissolution and vesicle formation. These states could be described as a series of equilibrium structures that took the forms of vesicles, open lamellar sheets, and mixed micelles. As detergent was added to an aqueous suspension of vesicles, the octyl glucoside partitioned into the vesicles with a partition coefficient of 63. This was accompanied by leakage of small molecules and vesicle swelling until the mole fraction of detergent in the vesicles was just under 50% (detergent:lipid ratio of 1:1). Near this point, a transition was observed by an increase in turbidity and release of large molecules like inulin, consistent with the opening of vesicles. Both a turbidity maximum and a sharp increase in fluorescence were observed at a detergent to lipid mole ratio of 2.1:1. This was interpreted as the lower boundary of a region where both lamellar sheets and micelles are at equilibrium. At a detergent:lipid ratio of 3.0:1, another sharp change in resonance energy transfer and clarification of the suspension were observed, demarcating the upper boundary of this two-phase region. This latter transition is commonly referred to as solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescent Dyes↗
PubMed1988

Family attitudes and patient social adjustment in a longitudinal study of outpatient schizophrenics receiving low-dose neuroleptics: the family's view.

Adverse effects of neuroleptic medication have led to the attempt to develop alternative strategies for the treatment of schizophrenia, but it is generally conceded that these strategies may have their own negative outcomes in the form of symptom exacerbation, reduced social performance and worsened family interactions. This paper examines the effect of one such strategy, low doses of medication, on the social adjustment of and family response to chronic schizophrenic outpatients. Patients who were randomly assigned to either a low-dose or standard-dose condition were rated by their families on various aspects of social adjustment. Despite a considerably higher relapse rate in the low-dose condition, families reported patients in the low-dose condition to be no poorer in their social adjustment than standard-dose patients. In addition, families of low-dose patients were more satisfied with their patients' overall level of adjustment and were no more rejecting at endpoint than families of standard-dose patients. Low-dose patients were viewed even more favorably when patients who relapsed were excluded from the analysis. Negative family attitudes, particularly rejection, measured at study entry, were found to predict time to relapse in the low-dose group. Implications for treatment and family intervention are discussed.

Adolescent↗
PubMed1987

Secretion of a growth inhibitory factor by ZR-75-1 human breast cancer cells.

Cultures of the human mammary carcinoma line ZR-75-1 secrete a growth inhibitory factor (GIF) that, when diluted, slows the growth of MDA-MB-231 and MCF-7 cells. Undiluted "conditioned" media prevents cell division from occurring in both human breast cancer lines. ZR-75-1 cells are unaffected by this factor. The amount of GIF in the culture media is related to the confluency of the ZR-75-1 cells. The activity of this GIF is not altered by DNAse or RNAse but is destroyed by heating or trypsin. Growth inhibition is 85-90% reversible if conditioned media is replaced with fresh media.

Breast Neoplasms↗
PubMed1987

pH-dependent fusion of vesicular stomatitis virus with Vero cells. Measurement by dequenching of octadecyl rhodamine fluorescence.

We have studied fusion between membranes of vesicular stomatitis virus (VSV) and Vero cells using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We could identify the two pathways of fusion by the kinetics of R18 dequenching, effects of inhibitors, temperature dependence, and dependence on osmotic pressure. Fusion at the plasma membrane began immediately after lowering the pH below 6 and showed an approximately exponential time course, whereas fusion via the endocytic pathway (pH 7.4) became apparent after a time delay of about 2 min. Fusion via the endocytic pathway was attenuated by treating cells with metabolic inhibitors and agents that raise the pH of the endocytic vesicle. A 10-fold excess of unlabeled virus arrested R18VSV entry via the endocytic pathway, whereas R18 dequenching below pH 6 (fusion at the plasma membrane) was not affected by the presence of unlabeled virus. The temperature dependence for fusion at pH 7.4 (in the endosome) was much steeper than that for fusion at pH 5.9 (with the plasma membrane). Fusion via the endocytic pathway was attenuated at hypo-osmotic pressures, whereas fusion at the plasma membrane was not affected by this treatment. The pH profile of Vero-VSV fusion at the plasma membrane, as measured by the dequenching method, paralleled that observed for VSV-induced cell-cell fusion. Fusion was blocked by adding neutralizing antibody to the Vero-VSV complexes. Activation of the fusion process by lowering the pH was reversible, in that the rate of fusion was arrested by raising the pH back to 7.4. The observation that pH-dependent fusion occurred at similar rates with fragments and with intact cells indicates that pH, voltage, or osmotic gradients are not required for viral fusion.

Animals↗