When vesicles composed of an equimolar mixture of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and cholesterol are treated with phospholipase C, phospholipid hydrolysis occurs ...without major changes in vesicle architecture. In the same way, addition of sphingomyelinase leads only to sphingomyelin cleavage. However, when both enzymes are added together, their joint hydrolytic activities give rise to leakage-free vesicle aggregation, lipid mixing, and aqueous contents mixing, i.e. vesicle fusion. The contribution of both enzymes is unequal, the main role of sphingomyelinase being the production of relatively large amounts of ceramide that will facilitate the lamellar-to-nonlamellar transition in the formation of the fusion pore, whereas phospholipase C provides mainly a localized, asymmetric, high concentration of diacylglycerol that constitutes the trigger for the fusion process. The lipidic end-products of both enzymes cooperate in destabilizing and fusing the membranes in a way that is never achieved through the action of any of the enzymes individually, nor by the products themselves when premixed with the other lipids during liposome preparation. Thus the enzymes appear to be coupled through their reaction products. This is the first observation of membrane fusion induced by the concerted activities of two enzymes. Besides, considering that both diacylglycerol and ceramide are important metabolites involved in cell signaling, it may also provide new ideas in the exploration of “cross-talk” phenomena between different signal transduction pathways.
Ectromelia virus (ECTV) is the causative agent of mousepox, a lethal disease of mice with similarities to human smallpox. Mousepox progression involves replication at the initial site of infection, ...usually the skin, followed by a rapid spread to the secondary replicative organs, spleen and liver, and finally a dissemination to the skin, where the typical rash associated with this and other orthopoxviral induced diseases appears. Case fatality rate is genetically determined and reaches up to 100% in susceptible mice strains. Like other poxviruses, ECTV encodes a number of proteins with immunomodulatory potential, whose role in mousepox progression remains largely undescribed. Amongst these is a secreted homologue of the cellular tumour necrosis factor receptor superfamily member CD30 which has been proposed to modulate a Th1 immune response in vivo.
To evaluate the contribution of viral CD30 (vCD30) to virus pathogenesis in the infected host, we have adapted a novel transient dominant method for the selection of recombinant ECTVs. Using this method, we have generated an ECTV vCD30 deletion mutant, its corresponding revertant control virus as well as a virus encoding the extracellular domain of murine CD30. These viruses contain no exogenous marker DNA sequences in their genomes, as opposed to other ECTVs reported up to date.
We show that the vCD30 is expressed as a secreted disulfide linked trimer and that the absence of vCD30 does not impair mousepox induced fatality in vivo. Replacement of vCD30 by a secreted version of mouse CD30 caused limited attenuation of ECTV. The recombinant viruses generated may be of use in the study of the role of the cellular CD30-CD30L interaction in the development of the immune response. The method developed might be useful for the construction of ECTV mutants for the study of additional genes.
Phosphatidylcholine phospholipase C (EC 3.1.4.3) from Bacillus cereus has been assayed with substrates in the form of large unilamellar vesicles. Phosphatidylcholine, phosphatidylethanolamine (also a ...substrate for the enzyme), sphingomyelin, and cholesterol have been mixed in various proportions, in binary, ternary, and quaternary mixtures. A lag period, followed by a burst of enzyme activity, has been found in all cases. The activity burst was always accompanied by an increase in turbidity of the vesicle suspension. Varying lipid compositions while keeping constant all the other parameters leads to a range of lag times extending over 2 orders of magnitude (from 0.13 to 38.0 min), and a similar variability is found in maximal enzyme rates (from 0.40 to 55.9 min-1). Meanwhile, the proportion of substrate that is hydrolyzed during the lag period remains relatively constant at 0.10% moles of total lipid, in agreement with the idea that enzyme activation is linked to vesicle aggregation through diacylglycerol-rich patches. Phosphatidylethanolamine and cholesterol enhance the enzyme activity in a dose-dependent way: they reduce the lag times and increase the maximal rates. The opposite is true of sphingomyelin. These lipids exert each its own peculiar effect, positive or negative, either alone or in combination, so that the susceptibility of a given mixture to the enzyme activity can be to some extent predicted from its composition. Phospholipase C activity is not directly influenced by the formation of nonlamellar structures. However, the presence of lipids with a tendency to form nonlamellar phases, such as phosphatidylethanolamine or cholesterol, stimulates the enzyme even under conditions at which purely lamellar phases exist. Conversely sphingomyelin, a well-known stabilizer of the lamellar phase, inhibits the enzyme. Thus phospholipase C appears to be regulated by the overall geometry and composition of the bilayer.
Ectromelia virus (ECTV) is the causative agent of mousepox, a lethal disease of mice with similarities to human smallpox. Mousepox progression involves replication at the initial site of infection, ...usually the skin, followed by a rapid spread to the secondary replicative organs, spleen and liver, and finally a dissemination to the skin, where the typical rash associated with this and other orthopoxviral induced diseases appears. Case fatality rate is genetically determined and reaches up to 100% in susceptible mice strains. Like other poxviruses, ECTV encodes a number of proteins with immunomodulatory potential, whose role in mousepox progression remains largely undescribed. Amongst these is a secreted homologue of the cellular tumour necrosis factor receptor superfamily member CD30 which has been proposed to modulate a Th1 immune response in vivo. To evaluate the contribution of viral CD30 (vCD30) to virus pathogenesis in the infected host, we have adapted a novel transient dominant method for the selection of recombinant ECTVs. Using this method, we have generated an ECTV vCD30 deletion mutant, its corresponding revertant control virus as well as a virus encoding the extracellular domain of murine CD30. These viruses contain no exogenous marker DNA sequences in their genomes, as opposed to other ECTVs reported up to date. We show that the vCD30 is expressed as a secreted disulfide linked trimer and that the absence of vCD30 does not impair mousepox induced fatality in vivo. Replacement of vCD30 by a secreted version of mouse CD30 caused limited attenuation of ECTV. The recombinant viruses generated may be of use in the study of the role of the cellular CD30-CD30L interaction in the development of the immune response. The method developed might be useful for the construction of ECTV mutants for the study of additional genes.
Previous studies demonstrated safety, immunogenicity and efficacy of DNA/modified vaccinia virus Ankara (MVA) prime/boost vaccines expressing tryparedoxin peroxidase (TRYP) andLeishmaniahomologue of ...the mammalian receptor for activated C kinase (LACK) againstLeishmania majorchallenge in mice, which was consistent with results from TRYP protein/adjuvant combinations in non-human primates. This study aimed to conduct safety and immunogenicity trials of these DNA/MVA vaccines in dogs, the natural reservoir host ofLeishmania infantum, followed-up for 4 months post-vaccination. In a cohort of 22 uninfected outbred dogs, blinded randomised administration of 1000μg (high dose) or 100μg (low dose) DNA prime (day 0) and 1x108pfu MVA boost (day 28) was shown to be safe and showed no clinical side effects. High dose DNA/MVA vaccinated TRYP dogs produced statistically higher mean levels of the type-1 pro-inflammatory cytokine IFN-γ than controls in whole blood assays (WBA) stimulated with the recombinant vaccine antigen TRYP, up to the final sampling at day 126, and in the absence of challenge withLeishmania. TRYP vaccinated dogs also demonstrated significantly higher TRYP-specific total IgG and IgG2 subtype titres than in controls, and positivein vivointradermal reactions at day 156 in the absence of natural infection, observed in 6/8 TRYP vaccinated dogs. No significant increases in IFN-γ in LACK-stimulated WBA, or in LACK-specific IgG levels, were detected in LACK vaccinated dogs compared to controls, and only 2/9 LACK vaccinated dogs demonstrated DTH responses at day 156. In all groups, IgG1 subclass responses and antigen-specific stimulation of IL-10 were similar to controls demonstrating an absence of Th2/Tregresponse, as expected in the absence ofin vivorestimulation or natural/experimental challenge withLeishmania. These collective results indicate significant antigen-specific type-1 responses andin vivomemory phase cellular immune responses, consistent with superior potential for protective vaccine immunogenicity of DNA/MVA TRYP over LACK.
Cryo-transmission electron microscopy has been applied to the study of the changes induced by phospholipase C on large unilamellar vesicles containing phosphatidylcholine, as well as to the action of ...sphingomyelinase on vesicles containing sphingomyelin. In both cases vesicle aggregation occurs as the earliest detectable phenomenon; later, each system behaves differently. Phospholipase C induces vesicle fusion through an intermediate consisting of aggregated and closely packed vesicles (the "honeycomb structure") that finally transforms into large spherical vesicles. The same honeycomb structure is also observed in the absence of enzyme when diacylglycerols are mixed with the other lipids in organic solution, before hydration. In this case the sample then evolves toward a cubic phase. The fact that the same honeycomb intermediate can lead to vesicle fusion (with enzyme-generated diacylglycerol) or to a cubic phase (when diacylglycerol is premixed with the lipids) is taken in support of the hypothesis according to which a highly curved lipid structure ("stalk") would act as a structural intermediate in membrane fusion. Sphingomyelinase produces complete leakage of vesicle aqueous contents and an increase in size of about one-third of the vesicles. A mechanism of vesicle opening and reassembling is proposed in this case.
Phospholipase C activity has been assayed with phosphatidylcholine as substrate in the presence of sodium cholate at concentrations well below those producing lipid solubilization. With short-chain ...phosphatidylcholine, which exists in monomeric form in aqueous solution, cholate has little or no effect. However, when the substrate is egg phosphatidylcholine in the form of bilayers, small cholate concentrations (below 1 mM, corresponding to an effective surfactant:lipid ratio below 0.05) increase the maximum enzyme rates by about threefold, while decreasing drastically the latency periods of enzyme activity. Previous studies from this laboratory have associated the phospholipase enhancing activity of a variety of amphiphiles to their ability to facilitate the formation of inverted hexagonal phospholipid structures, yet sodium cholate has the opposite effect, stabilizing the lamellar versus the inverted hexagonal phase. This suggests that cholate is activating phospholipase C through a hitherto undescribed mechanism. Sodium cholate concentrations above 1 mM decrease further the enzyme lag time, but they are less effective in enhancing enzyme rates. These observations may be pertinent in the analysis of biochemical data with purified lipases, as well as in physiological studies of biliary function.
Previous studies from this laboratory have shown that the enzymic generation of diacylglycerol in bilayers by phospholipase C may lead to membrane fusion through the formation of transient ...non-lamellar lipidic intermediates. The present paper intends to explore the correlations existing among the three main processes involved, namely (a) the induction (or inhibition) of lamellar-to-non-lamellar phase transitions in lipid mixtures through the addition of small (< 5 mol%) proportions of other lipids, (b) the promotion, by the latter lipids, of fusion in otherwise stable phospholipid vesicles (large unilamellar liposomes) under conditions leading to inverted hexagonal/inverted cubic phase formation in bulk lipid systems, and (c) the modulation, by the same small proportions of lipids, of phospholipase C hydrolysis of phosphatidylcholine in liposome bilayers. It is concluded that phospholipase C may give rise to non-lamellar lipidic structures that in turn permit liposomal fusion to occur, but neither enzyme activity is directly modulated by non-lamellar phase formation, nor will whatever kind of enzyme-induced non-lamellar structure give rise to fusion. Moreover, only under certain kinetic conditions will the enzyme give rise to the organization of non-lamellar structures that are conducive to the fusion event.
The coronavirus disease 2019 (COVID-19) pandemic has affected the world radically since 2020. Spain was one of the European countries with the highest incidence during the first wave. As a part of a ...consortium to monitor and study the evolution of the epidemic, we sequenced 2,170 samples, diagnosed mostly before lockdown measures. Here, we identified at least 500 introductions from multiple international sources and documented the early rise of two dominant Spanish epidemic clades (SECs), probably amplified by superspreading events. Both SECs were related closely to the initial Asian variants of SARS-CoV-2 and spread widely across Spain. We inferred a substantial reduction in the effective reproductive number of both SECs due to public-health interventions (R
< 1), also reflected in the replacement of SECs by a new variant over the summer of 2020. In summary, we reveal a notable difference in the initial genetic makeup of SARS-CoV-2 in Spain compared with other European countries and show evidence to support the effectiveness of lockdown measures in controlling virus spread, even for the most successful genetic variants.
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