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.     

Characterization of an endogenous substrate of the insulin receptor in cultured cells

Using antiphosphotyrosine antibodies, we have characterized the tyrosine phosphorylation of an endogenous substrate of the insulin receptor in Fao hepatoma cells and in Chinese hamster ovary cells transfected with a eukaryotic expression vector containing the human insulin receptor cDNA. In Fao cells, besides the beta-subunit of the insulin receptor, a protein with a molecular mass between 170 and 210 kDa designated pp185, undergoes tyrosine phosphorylation immediately after insulin stimulation reaching a maximum level within 30 s. After 4 h of continuous insulin stimulation, the labeling of pp185 decreased to less than half of its original intensity, whereas the insulin receptor was unchanged. After 24 h of insulin stimulation, the phosphotyrosine-containing insulin receptor decreased by 75% owing to down-regulation, whereas the pp185 was completely undetectable. By several biochemical and physiological criteria, the pp185 is distinct from the insulin receptor. The pp185 and the beta-subunit of the insulin receptor were strongly labeled with [32P]orthophosphate, but in contrast to the insulin receptor, the pp185 was not labeled by cross-linking with 125I-insulin or surface 125I iodination. Unlike the insulin receptor, the pp185 was extracted from Fao cells without detergent, and tryptic phosphopeptide mapping of the pp185 and the insulin receptor yielded distinct patterns. Thus, the pp185 is not located at the external face of the plasma membrane and does not bind insulin. Treatment of Fao cells with the phorbol ester, phorbol 12-myristate 13-acetate, stimulated the phosphorylation of two proteins with molecular weights of 170 and 210 kDa which were immunoprecipitated with the anti-phosphotyrosine antibody. Subsequent insulin stimulation increased the phosphorylation of the 210 kDa protein, but the pp185 was not detected. Increasing the concentration of the human insulin receptor in the Chinese hamster ovary cells by transfection with a plasmid containing the human insulin receptor cDNA caused a higher level of tyrosine phosphorylation of the beta-subunit and the pp185. These data support the notion that the insulin signal may be transmitted to a cellular substrate (pp185) which may initiate insulin action at intracellular sites.     

Kinetics of pH-dependent fusion between influenza virus and liposomes

The pH-dependent fusion between influenza virus and liposomes (large unilamellar vesicles) has been investigated as a model for the fusion step in the infectious entry of the virus into cells. Fusion was monitored continuously, with a fluorescence assay based on resonance energy transfer (RET) [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099], which allows an accurate quantitation of the fusion process. Evidence is presented indicating that the dilution of the RET probes from the liposomal bilayer into the viral membrane is not due to transfer of individual lipid molecules. The initial rate and final extent of the fusion reaction increase dramatically with decreasing pH, fusion being virtually complete within 1 min at pH 4.5-5.0. From experiments in which the ratio of virus to liposomes was varied, it is concluded that virus-liposome fusion products continue to fuse with liposomes, but not with virus. Fusion is most efficient with liposomes consisting of negatively charged phospholipids, while phosphatidylcholine and sphingomyelin are inhibitory. The reaction is completely blocked by an antiserum against the virus and inhibited by pretreatment of the virus with trypsin. The effect of proteolytic pretreatment at pH 7.4 is enhanced after preincubation of the virus at pH 5.0, consistent with the occurrence of a low pH induced, irreversible, conformational change in the viral fusion protein, the hemagglutinin (HA), exposing trypsin cleavage sites. When, after initiation of the fusion reaction at pH 5.0, the pH is readjusted to neutral, the process is arrested instantaneously, indicating that the low pH induced conformational change in the HA protein, in itself, is not sufficient to trigger fusion activity.     

Intermediates in influenza induced membrane fusion.

Our results show that the mechanism by which influenza virus fuses with target membranes involves sequential complex changes in the hemagglutinin (HA, the viral fusion protein) and in the contact site between virus and target membrane. To render individual steps amenable to study, we worked at 0 degree C which decreased the rate of fusion and increased the efficiency. The mechanism of fusion at 0 degree C and 37 degrees C was similar. The process began with a conformational change in HA which exposed the fusion peptides but did not lead to dissociation of the tops of the ectodomain of the trimer. The change in the protein led to immediate hydrophobic attachment of the virus to the target liposomes. Attachment was followed by a lag period (4-8 min at 0 degree C, 0.6-2 s at 37 degrees C) during which rearrangements occurred in the site of membrane contact between the virus and liposome. After a further series of changes the final bilayer merger took place. This final fusion event was not pH dependent. At 0 degree C efficient fusion occurred without dissociation of the top domains of the HA trimer, suggesting that a transient conformation of HA is responsible for fusion at physiological temperatures. The observations lead to a revised model for HA mediated fusion.     

Chiral recognition by 1H-NMR,EPR, and ENDOR spectroscopy

Chiral recognition with magnetic methods requires the formation of diastereomers. Due to the variety of appropriate reactions, hydrogen bond formation, esterification, and acetalization as well as host–guest interactions were chosen for basic investigations. The results obtained indicate that in the case of diamagnetic compounds the chemical shifts and for paramagnetic compounds the β-proton coupling constants are the most useful parameters. By combination of both pieces of information, assignment of the absolute configuration was achieved. © 1993 Wiley-Liss, Inc.     

Die V?gel des dauro-mandschurischen Uebergangsgebietes

Ohne Zusammenfassung     

SOME PROPERTIES OF SUSPENDED SEDIMENT ADSORBED CATIONS IN TURBID FRESHWATERS OF SOUTH AFRICA

SUMMARY

Large quantities of suspended sediments are common in many of South Africa's fresh-waters. Temporal and spatial variations in tile amounts of cations adsorbed were recorded. The adsorption appears to be dependent on valency, because greater quantities of the higher valencies are adsorbed. Ca⁺⁺ dominated the adsorbed cations and Mg the dissolved fraction. Water originating from the Beaufort Series contained high sodium concentrations. Fe⁺⁺ dominated the adsorbed minor cations. Large quantities of sediments transported by rivers enter impoundments. The adsorbed ions transported in this way are influenced by the type of suspended sediment and form a significant part of the total input of ions.