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Biomedical subjects

R Blumenthal

Publications and source records attributed to R Blumenthal.

At least 73 records · Page 4Linked to original sources
PubMed1993

Role of the fusion peptide sequence in initial stages of influenza hemagglutinin-induced cell fusion.

The fusion activity of influenza hemagglutinin (HA) and of HA proteins altered in the amino terminus of HA2 (fusion peptide) by site-directed mutagenesis (Gething, M.-J., Doms, R. W., York, D., and White, J. (1986) J. Cell Biol. 102, 11-23) was analyzed following expression in CV-1 cells using SV40-HA recombinant virus vectors. Fusion was monitored by the redistribution of lipid and cytoplasmic dyes between fluorescently labeled erythrocytes and HA-expressing CV-1 cells using spectrofluorometry and fluorescence microscopy. The kinetics of lipid redistribution after lowering the pH showed the same pattern for wild type HA and nonlethal mutants, although there were shifts in the pH threshold. The time for commitment to the fusogenic state and the temperature dependence of the processes leading to HA-mediated fusion were also the same for wild type and nonlethal mutants. However, striking differences were observed between wild type HA and the nonlethal mutants in their ability to induce pH-dependent redistribution from erythrocytes to HA-expressing cells of large molecular weight (M(r) > 10,000) fluorescently labeled dextran molecules. The data indicate that the kinetic processes which are measurable in the time range of seconds are insensitive to the structure of the fusion peptide. Surprisingly, however, the fusion peptide plays an important role in later processes related to pore widening which eventually results in delivery of the nucleocapsid into the cell.

Amino Acid Sequence↗
PubMed1993

Fusion of human B cell lines with HIV-1 envelope-expressing T cells is enhanced by antigen-specific Ig receptors. Possible mechanism for elimination of gp120-specific B cells in vivo.

The possible contribution of Ag-specific Ig receptors on B cells to syncytium formation with HIV-1 envelope (env)-expressing cells was examined. A unique model system was designed that used anti-TNP/TNP interactions between a panel of TNP-specific human B cell lines and TNP-haptenated HIV-1 env-expressing T cells. The prototype B cell line 1:13 (CD4dull) produced few syncytia with vaccinia gp120/41-infected CD4- T cell effectors. However, TNP-haptenation of the HIV-1 env-expressing cells resulted in a five- to 10-fold increase in syncytium formation. The "enhanced" syncytia were blocked by OKT4A mAb, soluble CD4, anti-TNP serum, and TNP-BSA, suggesting a role for both CD4 and Ig receptors. In contrast, the number of syncytia formed between CD4+ CEM T cells and TNP-haptenated effectors was reduced by 30 to 40%, compared with the unhaptenated effectors, suggesting that a fraction of the TNP haptens bound close to the CD4 binding regions on the gp120 envelope, which was confirmed by other experiments. The possibility that B cells specific for the CD4 binding site on HIV-1 gp120 may be involved in syncytium formation with HIV-1 env-expressing cells was tested by screening a panel of five hybrid B cell lines from HIV-1-seropositive individuals. One of these lines produced anti-gp120 antibodies, which bound near the CD4 binding site, and also formed syncytia with HIV-1 env-expressing cells. This study suggests that, in addition to CD4 receptors, certain B cell Ig receptors that bind to gp120 may induce conformational changes leading to cell fusion and their elimination.

B-Lymphocytes↗
PubMed1993

The block to HIV-1 envelope glycoprotein-mediated membrane fusion in animal cells expressing human CD4 can be overcome by a human cell component(s).

Membrane fusion mediated by interaction of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein with the human CD4 molecule generally requires that the CD4 be expressed on a human cell. The failure of murine or simian cells expressing human CD4 to form syncytia upon mixing with cells expressing envelope glycoprotein could not be corrected by expression of both molecules at extremely high surface levels using vaccinia virus expression vectors. Video fluorescence microscopic analysis of fluorescent dye transfer between fusing cells indicated that the block occurred at the level of membrane fusion between individual pairs of cells. To gain insight into the basis for this fusion block, we tested the ability of fluorescent probe cells expressing envelope glycoprotein to fuse with transient animal x human hybrid giant cells expressing human CD4. The hybrid giant cells were generated either by low-pH-induced fusion of vaccinia-infected cells or by CD4/HIV-1 envelope glycoprotein-mediated cell fusion. We observed that envelope glycoprotein-expressing probe cells efficiently fused with CD4-expressing animal x human hybrid giant cells, independent of whether the CD4 was originally expressed on the animal or on the human cell. Fusion did not occur with CD4-expressing giant cells derived from animal cells alone. These results indicate that the fusion block is not due to dominant inhibitory components in the animal cell. Rather, they suggest that human cells contain an additional component(s) which, when transferred to the CD4-bearing animal cell, confers the ability to undergo membrane fusion mediated by the HIV-1 envelope glycoprotein.

Animals↗
PubMed1993

A new approach to measure fusion activity of cloned viral envelope proteins: fluorescence dequenching of octadecylrhodamine-labeled plasma membrane vesicles fusing with cells expressing vesicular stomatitis virus glycoprotein.

Fusion between fluorescently labeled plasma membrane vesicles (PMV) and cells expressing vesicular stomatitis virus (VSV) glycoprotein (G-protein) was investigated by utilizing a lipid mixing assay based on fluorescence dequenching of octadecyl rhodamine (R18). The PMVs were prepared from Vero cells by hypotonic lysis. The G-protein was expressed on the cell surface either following infection with intact VSV or with an adenovirus vector (AdG12) containing the gene for the G-protein. Fusion was temperature and pH dependent and was inhibited by VSV G-antiserum. The pH dependence of PMV fusion paralleled that observed for VSV-cell fusion and VSV-induced syncytia formation. The kinetics of fusion followed an exponential dependence on time without an observable time lag after lowering pH. These findings indicate that dequenching R18-labeled PMV reliably represents the basic features of fusion of VSV with cells and can be used as a new tool in the study of fusion activity of virus envelope proteins expressed in cells.

Animals↗
PubMed1993

Percutaneous transluminal coronary angioplasty through 6F diagnostic catheters: a feasibility study.

This study evaluated the feasibility of performing coronary angioplasty through 6F diagnostic catheters by mainly using over-the-wire balloon systems on 84 lesions in 70 patients. Procedural variables, including vessel opacification and angioplasty outcome, were assessed. Changes in hematocrit after angioplasty were compared for 6F versus 7F and 8F systems. Successful 6F dilatation was performed in 72 (85.7%) of 84 lesions and 58 (82.9%) of 70 patients. Seven of the 12 lesions unable to be dilated with 6F systems were successfully dilated with larger French systems. Coronary artery opacification with the 6F catheters after balloon dilation was less than optimal with the balloon and guidewire still in the catheter. Changes in hematocrit after 6F procedures were significantly less than for 8F procedures (-2.1% vs -4.2%, respectively, p < 0.01) but not for 7F procedures (-2.4%, p = not significant). Potential cost savings for angioplasty with 6F diagnostic catheters could be significant. Thus angioplasty with over-the-wire balloon systems in which 6F nontapered diagnostic catheters are used can be performed safely and with less procedural blood loss than with 8F systems. Significant problems encountered with the current catheter design were poor vessel opacification after balloon dilation and difficulties with balloon retraction.

Angioplasty, Balloon, Coronary↗
PubMed1993

Enhancement of viral fusion by nonadsorbing polymers.

Nonadsorbing polymers such as dextran and poly(ethylene glycol) enhance binding as well as extents of fusion of influenza virus with erythrocytes. Kinetics and extent of viral membrane fusion were measured using an assay based on lipid mixing of a fluorescent dye. The effects of nonadsorbing polymers were in the concentration range from 0 to 10 wt%, far below the concentration required to overcome hydration repulsion forces. The enhancing effects were dependent on the molecular weight of nonadsorbing polymer, and only occurred at molecular weight > 1500; this links the phenomena we observe to the so-called "excluded volume effect" of nonadsorbing polymers. The time delay between triggering and the onset of influenza virus fusion was significantly reduced in the presence of nonadsorbing polymers. High molecular weight poly(ethylene glycol) also induced fusion of vesicular stomatitis virus with intact erythrocytes, which do not serve as target of vesicular stomatitis virus fusion in the absence of the polymer. The forces between membranes which determine rate-limiting processes in viral fusion and how they are affected by nonadsorbing polymers are discussed.

Adsorption↗
PubMed1993

Primary sequence, evolution, and repetitive elements of the Gallus gallus (chicken) beta-globin cluster.

The DNA sequence of the Gallus gallus (chicken) beta-globin cluster was completed and analyzed. This G + C-rich region is 23.7 kb in length and includes the rho-, beta H-, beta A-, and epsilon-globin genes, the enhancer found between the beta A and epsilon genes, and three upstream DNase I hypersensitive sites. The CpG dinucleotides are nonrandomly distributed, being present at an increased relative frequency near the promoters and upstream hypersensitive sites. The cluster has an unusually low TA dinucleotide frequency. The upstream hypersensitive sites (5'HS1, 5'HS2, and 5'HS3) contain DNA sequence motifs recognized by erythroid transcription factors. However, no significant sequence similarity was found among the upstream hypersensitive sites and the beta A/epsilon enhancer. The G. gallus upstream site sequences were not similar to the upstream sites of the mammalian globin clusters, probably due to the small size of the functional regions and large evolutionary distance between the classes. The avian cluster evolved by gene duplication from an ancestor beta-globin gene, first producing the epsilon and the rho/beta H/beta A ancestor genes, then the rho and the beta H/beta A ancestor genes, and finally the beta H- and beta A-globins. Four probable gene conversions can be documented: beta A to beta H, epsilon to beta H, and rho/epsilon (twice). The cluster shows a massive overrepresentation of a non-LTR retrotransposon, CR1, which accounts for 16% of the DNA. We suggest that the locus is a preferred site for CR1 insertion.

Amino Acid Sequence↗
PubMed1993

Mycoplasma fermentans (incognitus strain) cells are able to fuse with T lymphocytes.

Characteristics of the fusion of Mycoplasma fermentans (incognitus strain) with cultured lymphocytes were investigated. The rate and extent of fusion were monitored continuously in an assay that measured lipid mixing on the basis of dequenching of a fluorescent probe, octadecylrhodamine (R18), incorporated into mycoplasmas. Fusion of M. fermentans was detected with CD4+ (Molt-3) cells, CD4- (12E1) cells, and primary peripheral-blood lymphocytes. The level of fusion was relatively low (8%-12%). Detection of a similarly low level of fusion by fluorescence microscopy suggested the involvement of a specific lymphocyte subpopulation. After a short lag period, fusion at 37 degrees C proceeded exponentially for approximately 30 minutes and was virtually complete at 60 minutes. Throughout the process, lymphocytes remained intact. Fusion was stimulated by CaCl2 but not by MgCl2; its inhibition by antisera to M. fermentans and by pretreatment of M. fermentans with proteolytic enzymes implied that the mycoplasmas possessed a proteinase-sensitive receptor involved in fusion. Mycoplasmas were rendered nonfusogenic by treatment with the uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone; 5 microM) but were unaffected by treatment with chlorpromazine (10 microM) or DCCD (dicyclohexylcarbodiimide; 50 microM); these findings suggested that a proton gradient across the cell membrane is required for fusion.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Cell fusion mediated by interaction of a hybrid CD4.CD8 molecule with the human immunodeficiency virus type 1 envelope glycoprotein does occur after a long lag time.

Several domains of CD4 have been suggested to play a critical role in events that follow its binding to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41). It has been reported previously that cells expressing a chimeric molecule consisting of the first 177 residues of human CD4 attached to residues from the hinge, transmembrane, and cytoplasmic domains of human CD8 did not form syncytia with HIV-1-infected cells (L. Poulin, L.A. Evans, S. Tang, A. Barboza, H. Legg, D.R. Littman, and J.A. Levy, J. Virol. 65: 4893-4901, 1991). In contrast, we found that the hybrid CD4.CD8 molecule expressed in human cells did render them susceptible to fusion with cells expressing HIV-1IIIB or HIV-1RF envelope glycoproteins encoded by vaccinia virus recombinants, but only after long lag times. The lag time of membrane fusion mediated by the hybrid CD4.CD8 molecule was fivefold longer than that for the wild-type CD4 molecule. However, the rate of binding to and the affinity of soluble gp120 for membrane-associated CD4.CD8 were the same as for CD4. Both molecules were laterally mobile, as determined by patching experiments. Coexpression of the CD4.CD8 chimera with wild-type CD4 did not lead to interference in fusion but had an additive effect. Therefore, the proximal membrane domains of CD4 play an important role in determining the kinetics of postbinding events leading to membrane fusion. We hypothesize that the long lag time is due to the inability of the CD4.CD8-gp120-gp41 complex to undergo the rapid conformational changes which occur during the fusion mediated by wild-type CD4.

CD4 Antigens↗
PubMed1993

Calcium ions are required for cell fusion mediated by the CD4-human immunodeficiency virus type 1 envelope glycoprotein interaction.

Calcium ions are required for fusion of a wide variety of artificial and biological membranes. To examine the role of calcium ions for cell fusion mediated by interactions between CD4 and the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41), we used two experimental systems: (i) cells expressing gp120-gp41 and its receptor CD4, both encoded by recombinant vaccinia viruses, and (ii) chronically infected cells producing low levels of HIV-1. Fusion was measured by counting the number of syncytia and by monitoring the redistribution of fluorescence dyes by video microscopy. Syncytia did not form in solutions without calcium ions. Addition of calcium ions partially restored the formation of syncytia. EDTA and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] blocked syncytium formation in culture media containing calcium ions. Membrane fusion as monitored by fluorescence dye redistribution also required calcium ions. Cell fusion increased with an increase in calcium ion concentration from 100 microM to 10 mM but was not affected by magnesium ions in the concentration range from 0 to 30 mM. Fibrinogen and fibronectin did not promote fusion in the absence or presence of Ca2+. Binding of soluble CD4 to gp120-gp41-expressing cells was not affected by Ca2+ and Mg2+. We conclude that Ca2+ is involved in postbinding steps in cell fusion mediated by the CD4-HIV-1 envelope glycoprotein interaction.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Quantitation of human immunodeficiency virus type 1 infection kinetics.

Tissue culture infections of CD4-positive human T cells by human immunodeficiency virus type 1 (HIV-1) proceed in three stages: (i) a period following the initiation of an infection during which no detectable virus is produced; (ii) a phase in which a sharp increase followed by a peak of released progeny virions can be measured; and (iii) a final period when virus production declines. In this study, we have derived equations describing the kinetics of HIV-1 accumulation in cell culture supernatants during multiple rounds of infection. Our analyses indicated that the critical parameter affecting the kinetics of HIV-1 infection is the infection rate constant k = Inn/ti, where n is the number of infectious virions produced by one cell (about 10(2)) and ti is the time required for one complete cycle of virus infection (typically 3 to 4 days). Of particular note was our finding that the infectivity of HIV-1 during cell-to-cell transmission is 10(2) to 10(3) times greater than the infectivity of cell-free virus stocks, the inocula commonly used to initiate tissue culture infections. We also demonstrated that the slow infection kinetics of an HIV-1 tat mutant is not due to a longer replication time but reflects the small number of infectious particles produced per cycle.

Cell Aggregation↗
PubMed1993

Hemagglutinin-neuraminidase enhances F protein-mediated membrane fusion of reconstituted Sendai virus envelopes with cells.

Reconstituted Sendai virus envelopes containing both the fusion (F) protein and the hemagglutinin-neuraminidase (HN) (F,HN-virosomes) or only the F protein (F-virosomes) were prepared by solubilization of the intact virus with Triton X-100 followed by its removal by using SM2 Bio-Beads. Viral envelopes containing HN whose disulfide bonds were irreversibly reduced (HNred) were also prepared by treating the envelopes with dithiothreitol followed by dialysis (F,HNred-virosomes). Both F-virosomes and F,HNred-virosomes induced hemolysis of erythrocytes in the presence of wheat germ agglutinin, but the rates and extents were markedly lower than those for hemolysis induced by F,HN-virosomes. Using an assay based on the relief of self-quenching of a lipid probe incorporated in the Sendai virus envelopes, we demonstrate the fusion of both F,HN-virosomes and F-virosomes with cultured HepG2 cells containing the asialoglycoprotein receptor, which binds to a terminal galactose moiety of F. By desialylating the HepG2 cells, the entry mediated by HN-terminal sialic acid receptor interactions was bypassed. We show that both F-virosomes and F,HN-virosomes fuse with desialylated HepG2 cells, although the rate was two- to threefold higher if HN was included in the viral envelope. We also observed enhancement of fusion rates when both F and HN envelope proteins were attached to their specific receptors.

Animals↗
PubMed1993

Quality of Life Enjoyment and Satisfaction Questionnaire: a new measure.

The Quality of Life Enjoyment and Satisfaction Questionnaire (Q-LES-Q) is a self-report measure designed to enable investigators to easily obtain sensitive measures of the degree of enjoyment and satisfaction experienced by subjects in various areas of daily functioning. The summary scores were found to be reliable and valid measures of these dimensions in a group of depressed outpatients. The Q-LES-Q measures were related to, but not redundant with, measures of overall severity of illness or severity of depression within this sample. These findings suggest that the Q-LES-Q measures may be sensitive to important differences among depressed patients that are not detected by the measures usually employed.

Adolescent↗
PubMed1992

A long-lived state for influenza virus-erythrocyte complexes committed to fusion at neutral pH.

The low pH-induced fusion of influenza virus with intact erythrocyte plasma membranes is preceded by a delay time following pH reduction, that is itself pH- and temperature dependent. At 37 degrees C/pH 4.8, lipid mixing between virus and target membranes begins < 2 s after pH reduction, whereas at 4 degrees C/pH 4.8, fusion does not commence until > 10 min after pH reduction. We have found that within this time period at 4 degrees C, a population of virus acquires the capacity to subsequently undergo fusion with high efficiency at elevated temperatures and pH 7.4. Both the kinetics and the extent of this pH 7.4 fusion depend upon the time of pre-incubation at pH 4.8/4 degrees C. Incubation at pH 7.4/4 degrees C, following this pre-incubation does not result in fusion, but the capacity to fuse at pH 7.4/37 degrees C is retained for a time period exceeding 1 h. The longevity of this fusion committed state makes it amenable to biochemical and immunological analysis. We have shown that it is insensitive to dithiothreitol, neuraminidase and trypsin, but is incapacitated by thermolysin or protease K. We conclude that only the HA2 sub-unit of influenza haemagglutinin is a necessary protein component of later stages of the fusion pathway.

Animals↗
PubMed1992

Role of viral envelope sialic acid in membrane fusion mediated by the vesicular stomatitis virus envelope glycoprotein.

Fusion of vesicular stomatitis virus (VSV) with cells and liposomes before and after treatment with neuraminidase was studied using the R18 dequenching assay. Desialylation of VSV significantly enhanced the extent of fusion with Vero cells but affected neither the pH dependence nor the binding of VSV to Vero cells. The enhanced fusion of asialo-VSV was observed both at the plasma membrane as well as via the endocytic pathway. Both VSV and asialo-VSV fused with liposomes made of neutral phospholipid, but only asialo-VSV fused with liposomes containing a 1:1 mixture of neutral and negatively charged phospholipid. To examine factors which contribute to the extent of fusion, we analyzed the various activation and inactivation reactions that take place as a result of low-pH triggering of VSV prebound to the target membrane. Lag times for the onset of fusion were similar for VSV and asialo-VSV, indicating that desialylation did not affect the activation reactions. However, exposure of VSV bound to target membranes at pH 6.5 for 400 s led to considerable inactivation, whereas little inactivation was seen after desialylation of VSV. These results are analyzed in terms of a model which allows us to determine which components of the overall fusion process are dominated by viral envelope sialic acid.

Animals↗
PubMed1992

Dextran sulfate inhibits fusion of influenza virus and cells expressing influenza hemagglutinin with red blood cells.

The influence of dextran sulfate with molecular weights of 500,000 and 8000 on binding and fusion of influenza virus (X31 strain) and of cells expressing influenza hemagglutinin (GP4F) with red blood cells (RBC) was investigated by spectrofluorimetry using virus and RBC labeled with the fluorescent dye octadecyl rhodamine B (R18). There was no significant inhibition of binding of virus and GP4F cells to red blood cells by dextran sulfate, but the polymer strongly inhibited the low pH induced fusion. Virus-RBC fusion was completely blocked by the high molecular weight dextran sulfate at concentrations as low as 0.5 mg/ml. Inhibition of RBC-GP4F cell fusion by dextran sulfate in the same concentration range was not as pronounced but the effect was potentiated by Ca2+. The polymer was only inhibitory when added at early steps of the fusion reaction, but the pH-induced conformational change of the hemagglutinin was not affected by dextran sulfate as measured by its susceptibility to proteolytic digestion. Removal of dextran sulfate after low pH-requiring steps allowed the system to fuse at neutral pH indicating that the inhibitory effect requires the continuous presence of dextran sulfate during the fusion reaction.

3T3 Cells↗