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.     

Interaction of immunoglobulin-coupled liposomes with rat liver macrophages in vitro

The interaction between liposomes coated with covalently linked rabbit immunoglobulin (RbIg-liposomes), and rat liver macrophages (Kupffer cells) in monolayer culture was studied biochemically with radioactive tracers and morphologically by electron microscopy. The attachment of immunoglobulin (Ig) to liposomes caused a five-fold increase in liposome uptake by the Kupffer cells at 37 degrees C, in comparison with uncoated liposomes. The uptake was linear with time for at least 4 h and linear with liposome concentration up to a lipid concentration of 0.2 mM. At 4 degrees C uptake, probably representing cell surface-bound liposomes, was reduced to a level of approx. 20% of the 37 degrees C values. Involvement of the Fc receptor in the uptake process was indicated by the reduction of RbIg-liposome uptake by more than 75% as a result of preincubating the cells with heat-aggregated human or rabbit Ig at concentrations (less than 2 mg/ml) at which bovine serum albumin (BSA) had virtually no effect on uptake. At high concentrations (10-35 mg/ml), however, albumin also reduced liposome uptake significantly (20-30%), which suggests an interaction of the RbIg-liposomes with the Kupffer cells that is partially non-specific. RbIg-liposome uptake was dependent on the amount of RbIg coupled to the liposomes. Maximal uptake values were reached at about 200 micrograms RbIg/mumol liposomal lipid. Electron microscopic observations on cells incubated with horseradish peroxidase-containing RbIg-liposomes demonstrated massive accumulation of peroxidase reaction product in intracellular vacuoles, showing that the uptake observed by label association represents true internalization.     

Feeding responses to solanaceous allelochemicals by larvae of the tobacco hornworm, Manduca sexta

Feeding responses of the oligophagous tobacco hornworm to allelochemicals prevalent in their host plants were determined in food choice-tests using filter paper discs laced with a test solution or water (control). Six solanaceous alkaloids, tomatine, tomatidine, solanine, solanocapsine, atropine and nicotine, were tested and only tomatine and solanocapsine were found to influence preference behavior. Solanocapsine (5 mM) deters feeding whereas tomatine (1 mM) stimulates feeding slightly. No synergistic effect of either tomatine or tomatidine with sucrose was found.The responses to tomatine are affected by previous feeding experience. Tomatine slightly stimulates feeding in larvae reared on tomato (Lycopersicon esculentum), but slightly deters feeding in larvae reared on Jerusalem cherry (Solanum pseudocapsicum). Such induced preference is absent for the other alkaloids tested, which indicates that these alkaloids do not by themselves induce preferences for the plants containing them.The non-alkaloid allelochemicals, chlorogenic acid, rutin, and 2-tridecanone also influenced food choice behavior. Chlorogenic acid is slightly stimulatory at its natural concentration (1mM), but strongly deterrent at higher concentrations. Rutin stimulates feeding in a concentration-dependent manner. Its activity must be due to the glycosylated structure, because both the aglycone (quercetin) and the sugar moiety (rutinose) are neutral. Removal of the glucose part of rutin, as in quercitrin, results in feeding deterrent activity. 2-Tridecanone is neutral at its concentration in cultivated tomato (1 mM), but strongly deterrent and toxic at higher concentrations. Preference behavior is not affected by solanesol, GABA, and a mixture of host plant compounds stimulatory for anothe solanaceous-specific feeder, the Colorado potato beetle (Leptinotarsa decemlineata).We conclude that the prevalent solanaceous alkaloids and other allelochemicals tested do not play important roles in food selection of the tobacco hornworm, although some may make small contributions.

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Résumé Des experiences de choix de chenilles oligophages de M. sexta ont été réalisees avec des disques de papier filtre imbiles d'eau ou de solutions des substances allélochimiques dominantes dans les plantes consommées. Sur les six alcaloïdes de solanées examinés: tomatine, tomatidine, solanine, solanocapsine, atropine et nicotine, seuls la tomatine et la solanocapsine ont influé sur le choix; la solanocapsine (5 mM) empêche la prise de nourriture, tandis que la tomatine (1 mM) la stimule légèrement. Aucun effet synergique de la tomatine ou de la tomatidine n'a été observé en présence de sucrose.La réponse à la tomatine est modifiée par la prise de nourriture antérieure. Elle stimule légèrement l'alimentation de chenilles élevées sur tomates (Lycopersicon esculentum), mais dissuade légèrement les chenilles élevées sur Solanum pseudocapsicum. II n'y a pas d'action induite semblable avec les autres alcalïdes examinés, ce qui indique que ces alcaloïdes ne peuvent pas induire par eux-mêmes de préférences pour les plantes qui les contiennent.Des substances allélochimiques non-alcaloïdes: acide chlorogénique, rutine, et 2-tridécanone, influent aussi sur le comportement de choix alimentaire. L'acide chlorogénique est légèrement stimulant à sa concentration naturelle (1 mM), mais fortement dissuasif aux concentrations supérieures. La rutine stimule la prise de nourriture en fonction de sa concentration. Son activité doit être due à sa structure glucosylate, puisqu'aussi bien l'aglycone (quercitine) que la moiteé sucrée (rutinose) sont neutres. La suppression de la partie glucose de la rutine, comme dans le cas de la quercitine, a un effet dissuasif. A sa concentration dans la tomate cultivée (1 mM), le 2-tridécanone est neutre, mais il est fortement dissuasif et toxique à des concentrations supérieures.Le comportement de choix n'est pas modifié par le solanésol, le GABA, et par un mélange de composés végétaux stimulant un consommateur spécifique de solanées, comme le doryphore (Leptinotarsa decemlineata).Nous pouvons conclure que les principaux alcaloïdes et autres substances allélochimiques des solanées que nous avons examinés n'interviennent pas d'une façon importante, mais peuvent avoir une influence secondaire, dans les choix alimentaires de Manduca sexta.

     

Biliary lipid secretion in the rat. The uncoupling of biliary cholesterol and phospholipid secretion from bile acid secretion by sulfated glycolithocholic acid

Glycolithocholic acid and its sulfated derivative are major metabolites of the secondary bile acid lithocholic acid in man. Both compounds are known to induce cholestasis in experimental animals. We compared the effects of these endogenous hepatotoxins on bile production and biliary lipid composition in rats with chronic biliary drainage. The compounds were administered enterally at relatively low rates (5-50% of the rats' endogenous bile acid secretion in these experiments) to simulate enterohepatic circulation. Both compounds were substantially secreted into bile (more than 90% of dose); sulfated glycolithocholic acid unchanged and glycolithocholic acid after hepatic hydroxylation predominantly in the form of glyco-beta-muricholic acid (cf. Kuipers et al. (1986) Am. J. Physiol. 251, G189-G194). Neither glycolithocholic acid nor its sulfated derivative affected the biliary excretion of endogenous bile acids or bile flow in these experiments. In spite of this, phospholipid and cholesterol secretion were significantly reduced by sulfated glycolithocholic acid but were not altered by glycolithocholic acid. Phospholipid and cholesterol secretion rapidly decreased to 25 and 50% of their initial values, respectively, at biliary output rates of sulfated glycolithocholic acid up to 2 mumol/h, and did not further decrease when this output was increased to 6 mumol/h. Small unilamellar liposomes consisting of cholesterol, [Me-14C]choline-labeled phosphatidylcholine, phosphatidylserine and [3H]cholesteryl oleate in a 5:4:1:0.1 molar ratio were employed to label intrahepatic lipid pools. Administration of sulfated glycolithocholic acid slightly reduced bile acid synthesis from [3H]cholesteryl oleate, but significantly reduced the biliary secretion of [14C]phospholipid. Glycolithocholic acid did not affect the hepatic processing of liposomal lipids. It is concluded that sulfated glycolithocholic acid at low doses causes the uncoupling of biliary lipid secretion from that of bile acids, which might represent in initiating event in sulfated glycolithocholic acid hepatotoxicity.     

Cloning and sequencing of the gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough

Abstract The gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough (148 amino acid residues), the first flavoprotein for which a three-dimensional structure has been determined, was cloned with the use of two synthetic oligonucleotides, designed to recognize the coding sequence for amino acid residues 11–19 and 98–103, respectively. The two oligonucleotides were used to screen a library of 900 λ-clones of the D. vulgaris chromosome. A single clone, λFL1, reacting with both probes was isolated. The entire structural gene for flavodoxin is contained in the 15 kb insert of λFL1 as found by nucleic acid sequencing. The codon usage in the flavodoxin gene is strongly biased towards G or C in the third codon position. A table in which codon usage information from all genes of D. vulgaris sequenced to date is combined is presented and should facilitate further gene cloning with oligonucleotide probes.     

Kinetics of nitrite oxidation in two Nitrobacter species grown in nitrite-limited chemostats

The influence of growth rate, the presence of acetate and variation in the dissolved oxygen concentration on the kinetics of nitrite oxidation was studied in suspensions of intact cells of Nitrobacter winogradskyi and Nitrobacter hamburgensis. The cells were grown in nitrite-limited chemostats at different dilution rates under chemolithotrophic and mixotrophic conditions. Growth of N. hamburgensis in continuous culture was dependent on the presence of acetate. Acetate hardly affected the maximal nitrite oxidation rate per cell (V _max), but displayed a distinctly negative effect on the saturation constants for nitrite oxidation (K _m ) of both Nitrobacter species. This effect was reversible; when acetate was removed from the suspensions the K _m -values for nitrite oxidation returned to their original values. A reduction of the dissolved oxygen concentration from 100% to 18% air saturation slightly decreased the V _max of chemolithotrophically grown N. winogradskyi cells, whereas a 2.3 fold increase was observed with mixotrophically grown cells of N. hamburgensis. It is suggested that the large variation in K _m encountered in field samples could be due to this observed phenotypic variability. The V _max per cell is not a constant, but apparently is dependent on growth rate and environmental conditions. This implies that potential nitrite oxidation activity and numbers of cells are not necessarily related. Considering their kinetic characteristics, it is unlikely that N. hamburgensis is able to compete succesfully with N. winogradskyi for limiting amounts of nitrite under mixotrophic conditions. However, at reduced partial oxygen tensions, N. hamburgensis may become the better competitor.     

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.     

Effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of liposomes by Kupffer cells in culture

We investigated the effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of large unilamellar liposomes by rat Kupffer cells in maintenance culture. The phospholipid vesicles were labeled in the lipid moiety with phosphatidyl[14C]choline and contained [3H]inulin or [125I]iodoalbumin as nondegradable and degradable markers of the aqueous vesicle content, respectively. Cytochalasin B and dihydrocytochalasin B, inhibitors of microfilament function, reduced inert inulin label uptake by 75% maximally, but residual uptake was not followed by release of lipid degradation products from the cells. By contrast, colchicine, an inhibitor of microtubule assembly, reduced uptake of liposomal inulin by maximally 55% but could not inhibit release of lipid degradation products from the cells. It is concluded that the cytochalasins partly inhibit uptake but fully prevent the arrival of internalized liposomes in the lysosomal compartment, while the action of colchicine is to slow down the overall process of uptake and subsequent transportation to the lysosomes. Monensin reduced inulin uptake to an extent similar to that found with colchicine, but reversibly blocked degradation of liposomal lipid and encapsulated protein. The kinetics of degradation of liposomal constituents suggests that residual uptake in the presence of monensin represents accumulation in an intracellular compartment. Trifluoperazine did not affect binding, internalization or degradation of encapsulated protein at low concentration (6 microM), but completely inhibited release of liposomal lipid degradation products under these conditions. At intermediate concentration (14 microM), the drug also reduced the internalization, while a high concentration (22 microM) was required to inhibit protein degradation as well. We conclude that trifluoperazine has multiple sites of action in the uptake and processing of liposomal constituents by Kupffer cells.     

Continuous monitoring of lactate during exercise in humans using subcutaneous and transcutaneous microdialysis

We have evaluated the possibility of monitoring the plasma lactate concentration in human volunteers during cycle ergometer exercise using subcutaneous and transcutaneous microdialysis. In transcutaneous microdialysis, the relative increase in dialysate lactate concentration exceeded that of plasma lactate concentration by a factor of 6 during exercise due to exercise-induced lactate secretion in sweat. During exercise the subcutaneous microdialysis dialysate lactate concentration underestimated the plasma lactate concentration possibly due to diffusion limitation or adipose tissue lactate production. While it was demonstrated that microdialysis can be used for on-line lactate monitoring, neither subcutaneous nor transcutaneous dialysate lactate concentration were linearly related to the plasma lactate concentration during exercise, and it was found therefore that it was not possible to monitor directly plasma lactate concentration during exercise.     

Gas-chromatographic analysis of poly(3-hydroxyalkanoates) in bacteria

Summary The accuracy and reproducibility of the gas-chromatographic method for the analysis of PHB and PHA in whole cells of Alcaligenes eutrophus H16 and Pseudomonas putida KT2442 were determined. It was found that for analysis of PHA the methanolysis time in the assay had to be increased to 4 h. Accuracy of the PHB and PHA assay were 0.018 mg and 0.304 mg respectively, based on estimation of the measurement error.