Balancing immunity and tolerance: genetic footprint of natural selection in the transcriptional regulatory region of HLA-G.

Human leukocyte antigen-G (HLA-G) has well-recognized immunosuppressive properties modulating the activity of many immune system cells, and polymorphisms observed at the HLA-G 5' upstream regulatory region (5'URR) may influence gene transcriptional regulation. In this study, we characterized the sequence variation and haplotype structure of the HLA-G 5'URR in worldwide populations to investigate the evolutionary history of the HLA-G promoter and shed some light into the mechanisms that may underlie HLA-G expression control. A 1.4-kb region, encompassing the known HLA-G regulatory elements, was sequenced in three African populations from Senegal, Benin and Congo, and data were combined with those available in the literature, resulting in a total of 1411 individuals from 21 worldwide populations. High levels of nucleotide and haplotype diversities, excess of intermediate-frequency variants and reduced population differentiation were observed at this locus when compared with the background genomic variation. These features support a strong molecular signature of balancing selection at HLA-G 5'URR, probably as a result of the competing needs to maintain both a maternal-fetal immune tolerance and an efficient host immune response to invading pathogens during human evolution. An extended analysis of a 300-kb region surrounding HLA-G revealed that this region is not involved in a hitchhiking effect and may be the direct target of selection.

Worldwide genetic variation at the 3' untranslated region of the HLA-G gene: balancing selection influencing genetic diversity.

The HLA-G (human leukocyte antigen-G) molecule plays a pivotal role in immune tolerance by inhibiting different cell subsets involved in both innate and adaptive immunity. Besides its primary function in maintaining the maternal-fetal tolerance, HLA-G has been involved in a wide range of pathological conditions where it can be either favorable or detrimental to the patient, depending on the nature of the pathology. Although several studies have demonstrated the utmost importance of the 3' untranslated region (3'UTR) in the HLA-G expression profile, limited data exist on the sequence variability of this gene region in human populations. In this study, we characterized the genetic diversity and haplotype structure of the HLA-G 3'UTR by resequencing 444 individuals from three sub-Saharan African populations and retrieving data from the 1000 Genomes project and the literature. A total of 1936 individuals representing 21 worldwide populations were combined and jointly analyzed. Our data revealed a high level of nucleotide diversity, an excess of intermediate frequency variants and an extremely low population differentiation, strongly supporting a history of balancing selection at this locus. The 14-bp insertion/deletion polymorphism was further pointed out as the likely target of selection, emphasizing its potential role in the post-transcriptional regulation of HLA-G expression.

A homogenizing process of selection has maintained an "ultra-slow" acetylation NAT2 variant in humans.

N-Acetyltransferase 2 (NAT2) is an important enzyme involved in the metabolism of a wide spectrum of naturally occurring xenobiotics, including therapeutic drugs and common environmental carcinogens. Extensive polymorphism in NAT2 gives rise to a wide interindividual variation in acetylation capacity, which influences individual susceptibility to various drug-induced adverse reactions and cancers. Striking patterns of geographic differentiation have been described for the main slow acetylation variants of the NAT2 gene, suggesting the action of natural selection at this locus. In the present study, we took advantage of whole-genome sequence data available from the 1000 Genomes project to investigate the global patterns of population genetic differentiation at NAT2 and determine whether they are atypical compared with the remaining variation of the genome. The nonsynonymous substitution c.590G>A (rs1799930) defining the slow NAT2*6 haplotype cluster exhibited an unusually low FST value compared with the genome average (FST = 0.006, P = 0.016). It was indicated as the most likely target of a homogenizing process of selection promoting the same allelic variant in globally distributed populations. The rs1799930 A allele has been associated with the slowest acetylation capacity in vivo, and its substantial correlation with the subsistence strategy adopted by past human populations suggests that it may have conferred a selective advantage in populations shifting from foraging to agricultural and pastoral activities in the Neolithic period. Results of neutrality tests further supported an adaptive evolution of the NAT2 gene through either balancing selection or directional selection acting on multiple standing slow acetylation variants.

Giant vesicles as models to study the interactions between membranes and proteins.

The interaction between polypeptides and membranes is a fundamental aspect of cell biochemistry. Liposomes have been used in this context as in vitro systems to study such interactions. We present here the case of giant vesicles (GVs), which, due to their size (radius larger than 10 microns), mimic more closely the situation observed in cell membranes and furthermore permit to study protein-membrane interactions by direct optical monitoring. It is shown that GVs formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine by electroformation are permeable to certain low molecular weight molecules such as the nucleic acid dye YO-PRO-1 and fluorescein diphosphate whereas conventional liposomes (large or small unilamellar liposomes) are not. In addition, it is shown that non-membrane proteins, such as DNases or RNases, added to the selected GVs from the outside, are able to convert their substrate, which is strictly localized on the internal side of the membrane. This effect is only seen in GVs (also when they are removed from the original electroformation environment) and is absent in conventional liposomes. The fact that these effects are only present in GVs obtained by electroformation and not in conventional small liposomes is taken as an indication that certain physico-chemical properties of the bilayer are affected by the membrane curvature, although the mechanism underlying such differences could not be established as yet.

Entrapment of nucleic acids in liposomes.

The entrapment efficiency of three main methods used in the literature for the encapsulation of nucleic acids in liposomes were studied using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. In particular the reverse phase method, the dehydration/rehydration method, and the freeze/thawing method were compared to each other under standardised conditions, i.e. using in every case the same concentration of guest molecules (DNA, tRNA and ATP as low molecular weight analogue) and equally extruded liposomes. The percentage of entrapment strictly referred to the material localized inside the liposomes, i.e. particular care was devoted to ruling out the contribution of the nucleic acid material bound to the outer surface of the liposomes: this was eliminated by extensive enzymatic digestion prior to column chromatography. Depending on the conditions used, the percentage of the entrapped material varied between 10 and 54% of the initial amount. Further, the encapsulation efficiency was markedly affected by the salt concentration, by the size of liposomes, but to a lower degree by the molecular weight of the guest molecules. In general, we observed that the freeze/thawing encapsulation procedure was the most efficient one. In a second part of the work the freeze/thawing method was applied to encapsulate DNA (369 bp and 3368 bp, respectively) using liposomes obtained from POPC mixed with 1-10% charged cosurfactant, i.e. phosphatidylserine (PS) or didodecyldimethylammonium bromide (DDAB), respectively. Whereas PS had no significant effect, the entrapment efficiency went up to 60% in POPC/DDAB (97.5:2.5) liposomes. The large entrapment efficiency of DNA permits spectroscopic investigations of the DNA encapsulated in the water pool of the liposomes. UV absorption and circular dichroism spectra were practically the same as in water, indicating no appreciable perturbation of the electronic transitions or of the conformation of the entrapped biopolymer. This was in contrast to the DNA bound externally to the POPC/DDAB liposomes which showed significant spectral changes with respect to DNA dissolved in water.

Co-oligopeptides of glycine and aromatic amino acids with variable distance between the aromatic residues. IV. Spectroscopic properties of tryptophan-containing peptides in a cyclohexane phase.

L-Tryptophan, L-tryptophanylglycine, glycyl-L-tryptophan, glycyl-L-tryptophanylglycine and glycyl-L-tryptophanylglycylglycyl-L-tryptophanylglycine have been transferred from an aqueous solution (generally 0.1 M NaOH) to cyclohexane, using the quaternary ammonium salt trioctylmethyl ammonium chloride (NR+4Cl-, soluble in cyclohexane but not in water) as the transporting agent. The spectroscopic properties of L-tryptophan and tryptophan-containing peptides have been studied in the cyclohexane phase. With respect to the aqueous solutions, ultraviolet absorption spectra are characterized by a considerable red shift of the absorption maxima and by a hypochromicity of up to 10%. Fluorescence spectra generally show emission maxima which are characteristic of polar environments, accompanied by a significant enhancement of the quantum yield. CD spectra have also been investigated for all peptides and compared with those for aqueous systems reported in preceding publications. All these spectral changes cannot be attributed solely to the cyclohexane solvent effect. It is suggested that these anomalous spectral properties of the tryptophan-containing compounds in the cyclohexane-NR+4 solution are due to the influence the electrostatic field of the ion pair has on the indole chromophore. The possible implications of this finding for the spectroscopic properties of aromatic residues buried in the polar interior of proteins are discussed.

Enzyme-containing liposomes can endogenously produce membrane-constituting lipids.

BACKGROUND: 'Giant vesicles' are liposomes that have diameters of several micrometers. It is possible to microinject biochemicals into a single vesicle and follow the progress of a chemical reaction in real time by light microscopy. We have previously used this technique to inject phospholipase A2 into giant vesicles; the vesicles disappeared as their components were hydrolyzed. Here we investigate whether the lipid components of a vesicle can be synthesized inside it. RESULTS: Giant vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) and palmitoyl-CoA were prepared in a solution containing sn-glycerol-3-phosphate. Microinjection of the enzyme sn-glycerol-3-phosphate-acyltransferase into the vesicle catalyzes the in situ production of the lipid membrane precursor 1-palmitoyl-sn-glycerol-3-phosphate, which remains incorporated in the membrane. The altered membrane chemistry causes shrinkage of the vesicle and formation of smaller liposomes on the inner surface at the site of injection. Similar transformations were seen when the enzyme was added to the outside of the vesicle. CONCLUSIONS: We have used the first step of the 'salvage pathway' for synthesis of POPC to demonstrate that it is possible to localize the synthesis of a lipid membrane precursor inside a giant vesicle. In the future it may be possible to combine the necessary enzymes and substrates to carry out the reactions for a complete metabolic pathway within a liposome.

Polymerase chain reaction in liposomes.

BACKGROUND: Compartmentalization of biochemical reactions within a spherically closed bilayer is an important step in the molecular evolution of cells. Liposomes are the most suitable structures to model this kind of chemistry. We have used the polymerase chain reaction (PCR) to demonstrate that complex biochemical reactions such as DNA replication can be carried out inside these compartments. RESULTS: We describe the first example of DNA amplification by the PCR occurring inside liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or of a mixture of POPC and phosphatidylserine. We show that these liposomes are stable even under the high temperature conditions used for PCR. Although only a very small fraction of liposomes contains all eight different reagents together, a significant amount of DNA is produced which can be observed by polyacrylamide gel electrophoresis. CONCLUSIONS: This work shows that it is possible to carry out complex biochemical reactions within liposomes, which may be germane to the question of the origin of living cells. We have established the parameters and conditions that are critical for carrying out this complex reaction within the liposome compartment.

Microinjection into giant vesicles and light microscopy investigation of enzyme-mediated vesicle transformations.

BACKGROUND: 'Giant vesicles' have diameters of several micrometers and can be observed by light microscopy. Their size may allow manipulation of individual vesicles and direct observation of the progress of a chemical reaction in real time. We set out to test this possibility using enzymatic hydrolysis of vesicle components as a model system. RESULTS: We describe a novel micromanipulation technique that allows us to microinject femtoliter amounts of a reagent solution adjacent to or into giant vesicles with diameters ranging from 10 to 60 microm. The vesicle transformations can be monitored directly in real time by light microscopy and recorded by video analysis. Snake venom phospholipase A2 was added to vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, and the enzymatic hydrolysis of components of the lipid bilayer was observed over time. A specific effect on the targeted giant vesicle was seen and video recorded, while the neighbouring vesicles remained unaffected. Addition of the enzyme to the outside of a vesicle caused it to burst, whereas injection of the enzyme inside a vesicle resulted in a slow and constant decrease in its size, until it eventually disappeared from the resolution power of the light microscope. CONCLUSIONS: These results show that it is possible to micromanipulate an individual vesicle, and to follow visually the progress of an enzymatic reaction occurring on the vesicle bilayer over time.

Spectroscopic investigations of peptide 401 from bee venom.

The circular dichroism (CD) and 1 H-nmr properties of peptide 401, a bee venom component with 22 amino acid residues and two disulfide bridges, have been studied under a variety of conditions and compared with those of the structurally related octadecapeptide apamin. The major component of the relatively intense CD signal in the 200-230-nm region in both cases probably arises from the rigid asymmetric ring structures of the disulfide bridges. CD spectra are practically unaffected by pH (in the region 1-7), solvent (water, trifluoroethanol, dioxane/water mixtures), concentration of peptide, or additions of salt (guanidinium chloride, KCl). Temperature changes (in the range 20-59°C) have only a modest influence. For both apamin and peptide 401, reduction of the two disulfide bridges results in a dramatic change of the CD spectrum, which acquires the characteristic form of a random coil. Preliminary 1 H-nmr data are presented for both the reduced and the oxidized form. Several resonance peaks could be assigned on the basis of the theoretical random-coil spectrum. In the oxidized forms, six slowly exchangeable amide protons could be found in a spectrum taken at low pH, which are ascribed to intramolecular hydrogen bonds. Each of the four protons of the two histidine residues of peptide 401 appears as two distinct resonance peaks in the oxidized form but not in the reduced form. This is interpreted as arising from conformational heterogeneity of peptide 401.

A mechanical model for the half-of-the-sites reactivity of oligomeric enzymes.

We developed a model which is able to provide a rationalization of the half-of-the-sites reactivity of oligomeric enzymes. According to this model, a dimeric enzyme is considered as a system of two coupled oscilators, which are able to transmit energy (information) to each other via a weak elastic coupling. In such a system, the energy may fluctuate between the two coupled elements so as to accumulate in one of the two at a time, i.e., at one time a certain energy state will be present in only one of the two elements, if this energy state (protomer conformation) is relevant for the chemical reaction, the conditions of half-of-the-sites reactivity may be fulfilled. The limits, and possible generalization, of this model are discussed.

Phospholipid-based reverse micelles.

Physicochemical investigations on the aggregation of phospholipids (mainly phosphatidylcholines) in organic solvents are reviewed and compared with the aggregation behaviour of phospholipids in aqueous medium. In particular we review the data showing that phosphatidylcholines (lecithins) form reverse micellar structures in certain apolar solvents. In these systems not only low molecular weight compounds but also catalytically active enzymes and entire cells can be solubilized. In addition, highly viscous phosphatidylcholine gels can be obtained in organic solvents upon solubilizing a critical amount of water. Generally, phospholipid-based reverse micelles can be regarded as thermodynamically stable models for inverted micellar lipid structures possibly occurring in biological membranes.

Lecithin organogel as matrix for transdermal transport of drugs.

Organogels obtained by adding small amounts of water to a solution of lecithin in organic solvents were studied as matrices for the transdermal transport of drugs. Gels obtained from isopropyl palmitate and cyclooctane were used (molar ratios of water to lecithin of 3 and 12, respectively). Preliminarily histological studies showed that the gels have no harmful effect when applied to the skin for prolonged periods. Data relative to the stability of the organogels with time are also presented. Scopolamine and broxaterol were used as model drugs, and the transdermal experiments were done with a Franz diffusion cell and human skin obtained from plastic surgery. The transport rate of scopolamine obtained with the lecithin gels was about one order of magnitude higher than that obtained with an aqueous solution of the drug at the same concentration. In contrast, the transport rates of scopolamine obtained with the microemulsion solution prior to gelation (molar ratio of water to lecithin, 0) were not different from those obtained with the gel. The same variations in transport rates were observed for broxaterol, in which case the flux through the skin was directly proportional to the concentration of drug in the gel. At a concentration of broxaterol of 75 mg/mL in the donor gel, the flux was 47 micrograms.h-1.cm-2. Because preliminary results showed that transdermal transport is successful with amino acids and peptides also, it is concluded that lecithin gels may be efficient vehicles for the transdermal transport of various drugs.

Tumor cell growth inhibition by liposome-encapsulated aromatic polyamidines.

Apart from its antiproteinase activity, the aromatic polyamidine TAPP-Br [the bromo derivative of 1,3-di-(p-amidinophenoxy)-2,2-bis-(p-amidinophenoxymethyl)propane (TAPP-H)] is able to inhibit the in vitro growth of a variety of tumor cell lines, including human melanoma, and breast and kidney carcinoma. We have now shown that TAPP-Br can efficiently be encapsulated into egg phosphatidylcholine vesicles. When incorporated into these liposomes, the inhibitory effect of TAPP-Br is significantly enhanced compared with that of the free drug. Based on these promising results, a proposal is made for the delivery of this antiproliferative agent to tumor cells by using liposomes as the vehicle.

Autopoiesis with or without cognition: defining life at its edge.

This paper examines two questions related to autopoiesis as a theory for minimal life: (i) the relation between autopoiesis and cognition; and (ii) the question as to whether autopoiesis is the necessary and sufficient condition for life. First, we consider the concept of cognition in the spirit of Maturana and Varela: in contradistinction to the representationalistic point of view, cognition is construed as interaction between and mutual definition of a living unit and its environment. The most direct form of cognition for a cell is thus metabolism itself, which necessarily implies exchange with the environment and therefore a simultaneous coming to being for the organism and for the environment. A second level of cognition is recognized in the adaptation of the living unit to new foreign molecules, by way of a change in its metabolic pattern. We draw here an analogy with the ideas developed by Piaget, who recognizes in cognition the two distinct steps of assimilation and accommodation. While assimilation is the equivalent of uptake and exchange of usual metabolites, accommodation corresponds to biological adaptation, which in turn is the basis for evolution. By comparing a micro-organism with a vesicle that uptakes a precursor for its own self-reproduction, we arrive at the conclusion that (a) the very lowest level of cognition is the condition for life, and (b) the lowest level of cognition does not reduce to the lowest level of autopoiesis. As a consequence, autopoiesis alone is only a necessary, but not sufficient, condition for life. The broader consequences of this analysis of cognition for minimal living systems are considered.

1H-NMR of reverse micelles. I: The surfactant resonances as probes for the AOT/water/isooctane system.

1H-NMR spectra of Aerosol-OT (AOT) reverse micelles in isooctane are reported at various water contents and several temperatures. The resonances from the AOT protons near the polar head are completely assigned. The NMR parameters (chemical shift and linewidth) appear to be suitable probes for characterizing the physico-chemical state of micelles, in particular in order to define the range in which the system is fully homogeneous and transparent. The results correlate well with those obtained from optical density measurements. Lanthanide ions dissolved in the water phase selectively perturb the AOT resonances from the protons nearest to the polar head; conversely, non-polar shift reagents soluble in the organic phase do not cause appreciable effects on any of the observable signals.

1H-NMR of reverse micelles. II: Conformational studies of peptides and proteins in the AOT/water/isooctane system.

The interaction of AOT reverse micelles with Met-enkephalin, the pancreatic secretory trypsin inhibitor (PSTI) and the epidermal growth factor (EGF) is examined by NMR methods and the three systems are compared. While Met-enkephalin adopts a folded conformation, PSTI appears to become highly flexible, suggestive of a non-specific interaction with the micelles. On the other hand, the EGF spectrum shows that, although the main globular features of the protein are retained in the presence of AOT, the C-terminal fragment has to rearrange its conformation when put in contact with the micelle wall.