Functional reconstitution of influenza virus envelopes.

We have examined several procedures for the reconstitution of influenza virus envelopes, based on detergent removal from solubilized viral membranes. With octylglucoside, no functionally active virosomes are formed, irrespective of the rate of detergent removal: in the final preparation the viral spike proteins appear predominantly as rosettes. Protein incorporation in reconstituted vesicles is improved when a method based on reverse-phase evaporation of octylglucoside-solubilized viral membranes in an ether/water system is employed. However, the resulting vesicles do not fuse with biological membranes, but exhibit only a non-physiological fusion reaction with negatively charged liposomes. Functional reconstitution of viral envelopes is achieved after solubilization with octaethyleneglycol mono(n-dodecyl)ether (C12E8), and subsequent detergent removal with Bio-Beads SM-2. The spike protein molecules are quantitatively incorporated in a single population of virosomes of uniform buoyant density and appear on both sides of the membrane. The virosomes display hemagglutination activity and a strictly pH-dependent hemolytic activity. The virosomes fuse with erythrocyte ghosts, as revealed by a fluorescence resonance energy transfer assay. The rate and the pH dependence of fusion are essentially the same as those of the intact virus. The virosomes also fuse with cultured cells, either at the level of the endosomal membrane or directly with the cellular plasma membrane upon a brief exposure to low pH.     

c-myc gene expression and interleukin-2 receptor levels in cloned human CD2+,CD3+ and CD2+,CD3- lymphocytes

The levels of c-myc mRNA and interleukin-2 receptors (IL-2 Rec) were studied in human peripheral blood lymphocytes (PBL); mature CD2+,CD3+ T cell clones and CD2+,CD3- natural killer (NK) cell clones, and CD2+,CD3+ and CD2-,CD3- T lymphoma cell lines. A transient induction of the expression of c-myc and IL-2 Rec was observed in PBL after activation with phytohemagglutinin (PHA). Expression of c-myc and IL-2 Rec was also found in the CD2+,CD3+ and CD2+,CD3- clones. The CD2+,CD3+ showed higher levels of c-myc mRNA and IL-2 Rec than the CD2+,CD3- clones. In three T lymphoma cell lines constitutively high levels of c-myc mRNA but no IL-2 Rec were found. Only in JURKAT (CD2+,CD3+), c-myc mRNA levels could be further enhanced by PHA. These results suggest that in the presence of PHA, expression of c-myc and IL-2 Rec is induced via the CD3 receptor, and in the absence of PHA and/or the CD3 receptor alternative routes of induction are involved.     

New RNA-mediated reactions by yeast mitochondrial group I introns.

The group I self-splicing reaction is initiated by attack of a guanosine nucleotide at the 5' splice site of intron-containing precursor RNA. When precursor RNA containing a yeast mitochondrial group I intron is incubated in vitro under conditions of self-splicing, guanosine nucleotide attack can also occur at other positions: (i) the 3' splice site, resulting in formation of a 3' exon carrying an extra added guanosine nucleotide at its 5' end; (ii) the first phosphodiester bond in precursor RNA synthesized from the SP6 bacteriophage promoter, leading to substitution of the first 5'-guanosine by a guanosine nucleotide from the reaction mixture; (iii) the first phosphodiester bond in already excised intron RNA, resulting in exchange of the 5' terminal guanosine nucleotide for a guanosine nucleotide from the reaction mixture. An identical sequence motif (5'-GAA-3') occurs at the 3' splice site, the 5' end of SP6 precursor RNA and at the 5' end of excised intron RNA. We propose that the aberrant reactions can be explained by base-pairing of the GAA sequence to the Internal Guide Sequence. We suggest that these reactions are mediated by the same catalytic centre of the intron RNA that governs the normal splicing reactions.     

Neutrophil association and degradation of normal and acute-phase high-density lipoprotein 3.

The interaction of normal and acute-phase high-density lipoproteins of the subclass 3 (N-HDL3 and AP-HDL3) with human neutrophils and the accompanying degradation of HDL3 apolipoproteins have been studied in vitro. The chemical composition of normal and acute-phase HDL3 was similar except that serum amyloid A protein (apo-SAA) was a major apolipoprotein in AP-HDL3 (approx. 30% of total apolipoproteins). 125I-labelled AP-HDL3 was degraded 5-10 times faster than 125I-labelled N-HDL3 during incubation with neutrophils or neutrophil-conditioned medium. Apo-SAA, like apolipoprotein A-II (apo-A-II), was more susceptible than apolipoprotein A-I (apo-A-I) to the action of proteases released from the cells. The amounts of cell-associated AP-HDL3 apolipoproteins at saturation were up to 2.8 times greater than N-HDL3 apolipoproteins; while apo-A-I was the major cell-associated apolipoprotein when N-HDL3 was bound, apo-SAA constituted 80% of the apolipoproteins bound in the case of AP-HDL3. The associated intact apo-SAA was mostly surface-bound as it was accessible to the action of exogenous trypsin. alpha 1-Antitrypsin-resistant (alpha 1-AT-resistant) cellular degradation of AP-HDL3 apolipoproteins also occurred; experiments in which pulse-chase labelling was performed or lysosomotropic agents were used indicated that insignificant intracellular degradation occurred which points to the involvement of cell-surface proteases in this degradation.     

Binding of NAD+ by cholera toxin.

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.     

Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody.

Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s).     

The interaction in vivo of transferrin and asialotransferrin with liver cells.

Rat transferrin or asialotransferrin doubly radiolabelled with 59Fe and 125I was injected into rats. A determination of extrahepatic and hepatic uptake indicated that asialotransferrin delivers a higher fraction of the injected 59Fe to the liver than does transferrin. In order to determine in vivo the intrahepatic recognition sites for transferrin and asialotransferrin, the liver was subfractionated into parenchymal, endothelial and Kupffer cells by a low-temperature cell isolation procedure. High-affinity recognition of transferrin (competed for by an excess of unlabelled transferrin) is exerted by parenchymal cells as well as endothelial and Kupffer cells with a 10-fold higher association (expressed per mg of cell protein) to the latter cell types. In all three cell types iron delivery occurs, as concluded from the increase in cellular 59Fe/125I ratio at prolonged circulation times of transferrin. It can be calculated that parenchymal cells are responsible for 50-60% of the interaction of transferrin with the liver, 20-30% is associated with endothelial cells and about 20% with Kupffer cells. For asialotransferrin a higher fraction of the injected dose becomes associated with parenchymal cells as well as with endothelial and Kupffer cells. Competition experiments in vivo with various sugars indicated that the increased interaction of asialotransferrin with parenchymal cells is specifically inhibited by N-acetylgalactosamine whereas mannan specifically inhibits the increased interaction of asialotransferrin with endothelial and Kupffer cells. Recognition of asialotransferrin by galactose receptors from parenchymal cells or mannose receptors from endothelial and Kupffer cells is coupled to active 59Fe delivery to the cells. It is concluded that, as well as parenchymal cells, liver endothelial and Kupffer cells are also quantitatively important intrahepatic sites for transferrin and asialotransferrin metabolism, an interaction exerted by multiple recognition sites on the various cell types.     

Influence of N6-methylation of residue A(5) on the conformational behaviour of d(C-C-G-A-A-T-T-C-G-G) in solution studied by 1H-NMR spectroscopy. 2. The hairpin form

The solution conformations of the oligonucleotides d(C-C-G-A-A-T-T-C-G-G) and d(C-C-G-A-m6A-T-T-C-G-G) as a function of temperature and sample concentration were investigated by means of 1H-NMR spectroscopy. The NMR spectra revealed that, at certain combinations of temperature and low sample and salt concentration, both compounds exist as a B-DNA-type duplex slowly (on the 1H-NMR time scale) interconverting with a monomeric species. From chemical shift data and imino-proton spectra, it is concluded that the monomeric species consists of a mixture of a hairpin form in rapid equilibrium with the random-coil form. The double-helical stem of the hairpin is formed by the six terminal cytidine and guanine residues, whereas the four core residues, -A-(m6)A-T-T-, partake in the loop. Thermodynamic analysis of the chemical shift of the resonances of the monomeric species vs temperature profiles of the two decamers and mutual comparison of these profiles indicate the following: the influence of N6-methylation of residue A(5) upon the local structure of the hairpin must be small; methylation decreases the stability of the duplex relative to the monomeric species: the temperature at which the fraction duplex equals 0.5 was found to be 312 K for the parent compound and 305 K for the methylated decamer at 2 mM sample concentration; methylation does not significantly alter the stability of the hairpin form relative to the random coil form: the Tm of the hairp----n equilibrium random-coil equilibrium is 308 K for the parent compound and 306 K for the methylated decamer. A higher fraction hairpin-like structure for the N6-methylated compound is observed under identical conditions of temperature and sample concentration: at 300 K, 2 mM sample concentration, the fraction hairpin form is 0.12 for d(C-C-G-A-A-T-T-C-G-G) and 0.20 for d(C-C-G-A-m6A-T-T-C-G-G). This finding appears to be a consequence of the reduced stability of the methylated dimeric species relative to the monomeric species, and to depend upon the sodium-ion concentration: it becomes more pronounced under low-salt conditions.