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Biomedical subjects

A Loyter

Publications and source records attributed to A Loyter.

At least 73 records · Page 4Linked to original sources
PubMed1984

Membrane-bound antiviral antibodies as receptors for Sendai virions in receptor-depleted erythrocytes.

Anti-Sendai virus antibodies were covalently coupled to neuraminidase-treated human erythrocytes by the use of the bifunctional crosslinking reagents, N-succinimidyl-3-(2-pyridyldithio)propionate or succinimidyl-4-(p-maleimidophenyl)butyrate. Neuraminidase-treated erythrocytes bearing antibodies were able to bind Sendai virus particles, while treated erythrocytes lacking the antibodies failed to do so. Virus particles attached to erythrocyte membranes via the antibodies were able to cause hemolysis (virus-cell fusion) and promoted cell-cell fusion. Similar results were obtained when the antibodies were coupled to cat erythrocytes which lack receptors for Sendai virus particles. Reconstituted Sendai virus envelopes, similar to intact virus particles, were able to hemolyze and to induce fusion of neuraminidase-treated antibody-bearing erythrocytes. However, reconstituted envelopes containing inactive HN (hemagglutinin-neuraminidase) but active F (fusion) glycoproteins, despite attachment to antibody-bearing erythrocytes, failed to hemolyze or to induce cell-to-cell fusion. Fusion could be restored by insertion of an active HN glycoprotein into the membranes of the reconstituted envelopes. These results suggest that the HN glycoprotein, besides being the viral attachment protein, also participates in the membrane fusion process.

Antibodies, Viral↗
PubMed1984

Fusion between Sendai virus envelopes and biological membranes as monitored by energy transfer methods.

Chlorophyll a and chlorophyll b have been inserted into reconstituted envelopes of Sendai virus particles. Fluorescence measurements indicated a high efficiency of energy transfer between the two chlorophyll molecules due to their close proximity in the viral envelope. Fusion of reconstituted, pigmented virus envelopes with various biological cell membranes at 37 degrees C resulted in a significant decrease in the yield of energy transfer. Reduction in the efficiency of energy transfer was temperature and time dependent, and was also dependent upon the ratio between the reconstituted Sendai virus envelopes (donor) and recipient cells (acceptor). No reduction in the efficiency of energy transfer was observed when non-fusogenic, reconstituted viral envelopes were incubated with cell membranes.

Animals↗
PubMed1984

Construction of fusogenic vesicles bearing specific antibodies. Targeting of reconstituted Sendai virus envelopes towards neuraminidase-treated human erythrocytes.

The cross-linking reagents succinimidyl-4-(p-maleimidophenyl)-butyrate and N-succinimidyl-3-(2-pyridyldithio)-propionate were used to covalently attach antibodies against human erythrocytes to the thiol-containing paraffin, dodecanethiol. The complex formed, dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody was inserted into the membranes of reconstituted Sendai virus envelopes. This was achieved by addition of the dodecanethiol-maleimidophenylbutyrate-antibody to a detergent solution (Triton X-100) containing the viral envelope phospholipids and glycoproteins. Removal of the detergent led to the formation of vesicles containing the viral glycoprotein and the dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody complexes within the same membrane. Reconstituted Sendai virus envelope-bearing antibodies against human erythrocytes were able to fuse with human erythrocytes (as was reflected by reconstituted Sendai virus envelope-induced hemolysis) from which the natural virus receptors were removed by treatment with neuraminidase. Thus, it appears that anti-human erythrocyte antibodies could substitute for the viral binding protein (hemagglutinin/neuraminidase glycoprotein) in mediating functional binding of the virus particles to the cell plasma membranes. Furthermore, from the results of the present work, it may be inferred that in addition to being the viral-binding protein, hemagglutinin/neuraminidase glycoprotein actively participates in the process of virus-cell fusion.

Antibodies↗
PubMed1984

A new method for reconstitution of highly fusogenic Sendai virus envelopes.

A new way for reconstituting highly fusogenic Sendai virus envelopes is described. As opposed to previously described methods, in the present one the detergent (Triton X-100) is removed by direct addition of SM-2 Bio-beads to the detergent solubilized mixture of the viral phospholipids and glycoproteins, thus avoiding the long dialysis step. The vesicles obtained in the present work resemble, in their composition, size and features, envelopes of intact Sendai virus particles. The present method allows the enclosure of low and high molecular weight material within the reconstituted viral envelopes.

DNA, Viral↗
PubMed1984

The use of specific antibodies to mediate fusion between Sendai virus envelopes and living cells.

Incubation of Sendai virus particles with non-ionic detergents such as Triton X-100 completely solubilizes the viral envelopes. Removal of the detergent from the supernatant (which contains the two main viral glycoproteins) leads to the formation of fusogenic, reconstituted viral envelopes. Soluble macromolecules such as DNA or proteins can be enclosed within the reconstituted vesicles, while membrane components can be inserted into the viral envelopes. Fusion of such loaded or 'hybrid' reconstituted envelopes with living cells in culture results in either microinjection or transfer of the viral components to the recipient cells. Thus such reconstituted envelopes can serve as efficient carriers for the introduction of macromolecules of biological interest into living cells in culture. A more specific vehicle has been constructed by chemically coupling anti-cell membrane antibodies (anti-human erythrocyte antibody) to the viral envelope. Such antibody-bearing intact virus particles or reconstituted envelopes bound to and fused with virus receptor-depleted cells. In addition, anti-Sendai virus antibodies were coupled to neuraminidase-treated human erythrocytes. Such antibodies mediated the binding and fusion of intact Sendai virus particles and their reconstituted envelopes to virus receptor-depleted cells.

Antibodies↗
PubMed1984

Attachment of Sendai virus particles to cell membranes: dissociation of adsorbed virus particles with dithiothreitol.

Sendai virus particles bind to human erythrocytes at 4 degrees C and fuse with them at 37 degrees C. The present work describes a new method by which adsorbed virus particles can be removed from human erythrocytes, allowing quantitative determination of the number of virus particles which can bind and fuse with human erythrocyte membranes. Through the use of 125I-labeled Sendai virus particles, it is shown that incubation with 50 mM dithiothreitol removed about 90 to 95% of adsorbed virus particles. Fused virus particles were resistant to treatment with dithiothreitol. Negligible amounts of 125I-labeled Sendai virus particles were removed by treatment with dithiothreitol after incubation of virus-cell complexes at 37 degrees C. Trypsinized virus particles were able to attach to, but not fuse with, human erythrocytes even after prolonged incubation at 37 degrees C. Treatment with dithiothreitol removed as much as 80 to 85% of trypsinized virus particles incubated with human erythrocytes at 37 degrees C. A quantitative determination revealed that about 1,000 to 1,200 and 600 to 800 Sendai virus particles can bind to or fuse with human erythrocytes, respectively.

Adsorption↗
PubMed1983

A possible involvement of virus-associated protease in the fusion of Sendai virus envelopes with human erythrocytes.

A proteolytic activity is shown to be associated with relatively purified preparations of intact Sendai virus particles or with their reconstituted envelopes which are vesicles containing mainly the viral glycoproteins. Intact Sendai virus as well as reconstituted Sendai virus envelopes have been shown to be able to hydrolyze various protein molecules such as the human erythrocyte membrane polypeptide designated as band 3 and soluble polypeptides such as histone and insulin B-chain. The results of the present work raise the possibility that a direct correlation exists between the virus-associated proteolytic activity and the ability of the virions to lyse cells, to fuse with their membranes, and to promote cell-cell fusion. Inhibitors of proteolytic enzymes such as phenylmethylsulfonyl fluoride, tosyllysinechloromethylketone and tosylamidephenylethylchloromethylketone, or combinations thereof, inhibit the virus-associated proteolytic activity concomitantly with inhibition of its hemolytic and fusogenic activities. Electron microscopic studies showed that the various inhibitors did not affect the binding ability of the virus preparations. The possible involvement of a protease in the process of virus-membrane fusion is discussed.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1983

Fusion-mediated injection of SV40-DNA. Introduction of SV40-DNA into tissue culture cells by the use of DNA-loaded reconstituted Sendai virus envelopes.

Sendai virus envelopes can be solubilized by non-ionic detergents such as Triton X-100. Removal of the detergent from a supernatant containing the solubilized viral envelope glycoproteins results in the formation of reconstituted fusogenic viral envelopes. When SV40-DNA is added to the reconstitution system, it is trapped within the viral envelope. Incubation of SV40-DNA-loaded Sendai virus envelopes with permissive cells (CV1 and TC7 cells) resulted in fusion-mediated injection of the trapped DNA, as was demonstrated by the ability of the injected cells to synthesize SV40-T-antigen. Quantitative estimation revealed that up to 20% of the injected cells were able to synthesize T-antigen. Loaded viral envelopes were able to inject SV40-DNA and to promote synthesis of T-antigen also in cells which are resistant to infection by intact SV40 viruses, such as F1' 1-4 cells. In addition, it is shown that reconstituted envelopes of Sendai virus are able to transfer membrane fragments from SV40 receptor-positive into SV40 receptor-negative cells, such as F1' 1-4 cells. After implantation of SV40 receptors, the F1' 1-4 cells became susceptible to infection by intact SV40 viruses.

Animals↗
PubMed1983

Fusion-mediated implantation of band 3 into living cells. A new system to study degradation of membrane proteins.

Sendai virus (SV) glycoproteins were co-reconstituted with the erythrocyte membrane polypeptide designated as band 3 (B3). Fusion of the hybrid vesicles, SV-B3, with L, HeLa and Friend erythroleukemic (FEL) cells led to fusion-mediated implantation of the erythrocyte band 3 into the plasma membranes of the above cells. By the use of radiolabelled band 3 [( 3H]H2DIDS-B3) it was possible to demonstrate that about 1-4 X 10(6) molecules of band 3 can be transferred to the membranes of each of the above cell lines. Cultivation of the recipient cells in growth medium resulted in gradual decrease of the cell-associated counts. The reduction pattern followed first-order kinetics, reaching t 1/2 of disappearance at about 25-30 h in the case of L and HeLa cells, and 57 h in the case of FEL cells. NH4Cl added to the growth medium significantly inhibited the disappearance of band 3 during growth. The studies of the present work indicate that the reduction in the cell-associated band 3 during growth may represent degradation of this polypeptide by the recipient cells.

Ammonium Chloride↗
PubMed1983

Restoration of Ca2+ influx and degranulation capacity of variant RBL-2H3 cells upon implantation of isolated cromolyn binding protein.

Recently, variants of the rat basophilic leukemia cells (RBL-2H3), deficient in their binding capacity for the antiallergic drug cromolyn but displaying unimpaired ability to bind IgE, were selected and cloned [Mazurek, N., Bashkin, P., Petrank, A. & Pecht, I. (1983) Nature (London) 303, 528-530]. Although the histamine content and the number of IgE receptors in these variants are similar to those of the parental cells, they cannot be stimulated immunologically to allow Ca(2+) influx and to degranulate. In contrast, the Ca(2+) ionophore A23187 causes these variants to degranulate, indicating that the mechanism distal to the Ca(2+) gating is intact in the variants. The cromolyn binding protein (CBP), present in the membranes of RBL-2H3 cells, has recently been isolated by affinity chromatography under nondenaturing conditions. In the current study we have used Sendaivirus envelopes as fusogenic carriers to implant the purified CBP into the membrane of variant basophils that were defective in it. This fusion leads to the restoration of Ca(2+) uptake and degranulation capacity of the variants after IgE-mediated stimulation. These restored activities seem to show a sigmoidal dependence on the amount of incorporated CBP. Saturation values comparable to those of the parental line are reached when the level of implanted CBP approaches its density on the latter line. The restored capacity is due to the implanted CBP, because the reinstated immunological response can be blocked by the inhibitory drug cromolyn and by monoclonal antibodies specific to CBP, both shown to prevent Ca(2+) uptake and degranulation in mast cells and parental RBL-2H3 cells. These results point out that CBP plays an important role in the Ca(2+) gating process resulting in degranulation.

Animals↗
PubMed1982

Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes.

Coprecipitation of DNA with calcium phosphate is a commonly used method of gene transfer in mammalian cells. We have found that DNA forms a tight complex with Ca Pi and that DNA in this complex is resistant to nucleases present in serum or added externally. Under optimal conditions, virtually all of the recipient mouse Ltk- Aprt- cells take up Ca Pi--DNA complexes, as determined by fluorescent dyes specific for DNA (4',6-diaminilo-2-phenylindol dihydrochloride) or for calcium salts (chlorotetracycline). However, only a small proportion of the cells have detectable CA Pi--DNA complexes in the nucleus. Uptake of the Ca Pi--DNA complexes was highly dependent upon the pH at which the Ca Pi--DNA complexes was formed and upon the concentration of DNA in the complex.

Animals↗
PubMed1980

Utilization of membranous lipid substrates by membranous enzymes. Hydrolysis of sphingomyelin in erythrocyte 'ghosts' and liposomes by the membranous sphingomyelinase of chicken erythrocyte 'ghosts'.

Incubation at 37 degrees C of haemolysed chicken erythrocytes ('chicken erythrocyte ghosts') resulted in hydrolysis of the membrane sphingomyelin, suggesting an activation of a latent sphingomyelinase during the haemolysis procedure. When this incubation was continued for several hours, the entire sphingomyelin of the erythrocyte 'ghosts' was hydrolysed and membranes were obtained that were devoid of sphingomyelin, but had an active sphingomyelinase. Mixing the latter membranes with human erythrocyte 'ghosts' or positively charged liposomes led to hydrolysis of the sphingomyelin in these two membranes. This suggested that, after haemolysis, the activated sphingomyelinase in the membrane of the chicken erythrocyte 'ghosts' could hydrolyse sphingomyelin in its own membrane ('intramembrane utilization') or adjacent membranes ('intermembrane utilization').

Animals↗