Lysozyme effect on oleic acid/oleate vesicles.

We have analysed by means of turbidimetric, dynamic light scattering (DLS), and fluorimetric techniques the effect of lysozyme on negatively charged oleic acid/oleate vesicles. The addition of lysozyme brings about a decrease in optical density of the vesicle population, which finally results in a size distribution of oleate vesicles shifted toward smaller mean diameters. On the contrary, (a) when phosphatidylserine vesicles were used, lysozyme induces an increase of turbidity and a shift toward larger vesicle sizes; and (b) the addition of histone H1 or poly-L-lysine produces an aggregative behavior both in oleate and in phosphatidylserine vesicles. Experiments carried out with calcein-containing vesicles indicate that the observed changes in the lysozyme/oleate system occur with partial leakage of the vesicle content. All this is taken to suggest that the interaction between lysozyme and oleate vesicles is of quite specific nature, and certainly not just due to electrostatic interactions.

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.

The use of liposomes for constructing cell models.

We illustrate here in a form of a short review some of the work developed in our and other groups aiming at performing inside liposomes enzymatic reactions relevant for the origin of life. The work on giant vesicles will not be considered here. The long-range goal of our work with SUVs or LUVs (small unilamellar vesicles or large unilamellar vesicles) is the construction of a model minimal cell. By this we mean a cell-like system containing the minimal and sufficient number of macromolecular components for expressingsome of the basic functions of a living cell- such as protein biosynthesis, growth and self-reproduction, homeostasis based on a primitive metabolism. We begin describing a POPC liposomal system containing some of the enzymes of the salvage cycle for the synthesis of lecithin; then vesicles containing the nucleotide phosphorylase enzyme for the polymerisation of ADP into poly(A); an oleate self-reproducing vesicular system which hosts Qß replicase for the replication of a RNA template; a POPC systems (POPC = 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine) hosting the elements for a polymerase chain reaction; and finally the attempts to organize inside liposomes the ribosomal system capable of the synthesis of poly(phenylalanine). This analysis of published work will be followed by the description of novel work aimed at expressing a protein (green fluorescent protein) inside liposomes. The possible development of this work and its limits will be discussed.

Molecular dynamics simulation of n-dodecyl phosphate aggregate structures.

Aggregates of n-dodecyl phosphate present an attractive model system of simple phospholipid amphiphile supramolecular structures for study by molecular dynamics simulation, since these systems have previously been studied experimentally under various conditions. A detailed molecular dynamics description of the properties of planar bilayer membranes (as a model for unilamellar vesicular membranes) and spherical micelles under various simulated conditions is presented. It is shown that the united-atom model of GROMOS96 applying the force-field parameter set 43A2 for biomolecular systems yields properties in agreement with experimental ones in most cases. Hydrogen bonding plays a role in stabilizing the bilayer aggregates at low pH, but not for the micelles, which are energetically favoured at high pH. NMR -S(CD) order parameters for a lipid bilayer system, the diffusion of amphiphiles within aggregates and of counterions, and lifetimes of hydrogen bonds between amphiphiles and to water are estimated from the MD simulations.

Enzyme immobilization in silica-hardened organogels.

In this study we describe a novel method for immobilizing enzymes in a solid nanocomposite matrix based on gelatin gels, which are subsequently hardened by in situ polymerization of tetraethoxysilane (TEOS). Chromobacterium viscosum lipase is taken as the example. This immobilization method possesses the advantages of enzyme entrapment in microemulsions, together with newly beneficial qualities, such as transparency, which permits direct spectroscopic investigation, and considerable mechanical stability in both aqueous and organic solvents, which results in the maintenance of enzymatic activity for several months. The first step is enzyme solubilization in AOT reverse micelles, followed by transformation of this solution into an organogel by the addition of gelatin. The enzyme-containing gel, is then hardened by the formation of silicate polymer. A glassy nanocomposite is obtained, which is optically transparent, so that the protein can be studied directly spectroscopically. Circular dichroic spectra of cytochrome-c are shown as an example. The nanocomposite material can be dried and ground, yielding a powder that is stable in both aqueous and organic solvents. After extensive washing with water, the enzyme-containing nanocomposite showed good activity in cyclohexane. The synthesis of water-insoluble fatty acid esters was carried out in this solvent with yields close to 90%. In this case, the enzyme preparations can be used over a period of several months without loss of activity or chemical yield.

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.

Liposome-assisted selective polycondensation of alpha-amino acids and peptides.

The main question of this paper is whether and to what extend lipid bilayers can aid in the polycondensation of amino acids and peptides. This means in particular how such bilayers can favor the selection of certain sequences out of a large number of theoretical possible ones. In a first series of experiments we started from a library of Trp-containing dipeptides of the type Trp-X where X is an amino acid residue; and we could show that, when adding this mixture to the POPC liposomes containing a hydrophobic quinoline condensing agent (EEDQ), only the hydrophobic Trp-Trp dipeptide is selected out by the liposomes and transformed into a longer oligomer. Trp-oligomers up to 29 monomers long (water insoluble) could be obtained by using the matrix support of liposomes. Mixed POPC/DDAB liposomes (positive charge) were used to produce co-oligopeptides that contain Trp and Glu residues in the same sequence. Arg/Trp and His/Trp containing sequences were obtained in presence of negatively charged liposomes (mixed POPC/DOPA-liposomes). The polycondensation of racemic NCA-amino acids has been studied to clarify if homochiral sequences are produced preferentially in presence or absence of liposomes. LC-MS and isotope labeling of the L-amino acid, participating in the polymerization reaction achieved this on the level of a direct product analysis. So the individual stereoisomer distribution up to a polymerization degree of 10 (in the case of Trp) could be determined. The data for Trp and other amino acids (Leu, Ile) and amino acid mixtures (Trp/Leu, Trp/Ile, Leu/Ile and Trp/Leu/Ile) show that homochiral sequences are produced preferentially if compared with a random (Bernoulli) distribution.

Protein expression in liposomes.

Compartmentalization is one of the key steps in the evolution of cellular structures and, so far, only few attempts have been made to model this kind of "compartmentalized chemistry" using liposomes. The present work shows that even such complex reactions as the ribosomal synthesis of polypeptides can be carried out in liposomes. A method is described for incorporating into 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC) liposomes the ribosomal complex together with the other components necessary for protein expression. Synthesis of poly(Phe) in the liposomes is monitored by trichloroacetic acid of the (14)C-labelled products. Control experiments carried out in the absence of one of the ribosomal subunits show by contrast no significant polypeptide expression. This methodology opens up the possibility of using liposomes as minimal cell bioreactors with growing degree of synthetic complexity, which may be relevant for the field of origin of life as well as for biotechnological applications.

About various definitions of life.

The old question of a definition of minimal life is taken up again at the aim of providing a forum for an updated discussion. Briefly discussed are the reasons why such an attempt has previously encountered scepticism, and why such an attempt should be renewed at this stage of the inquiry on the origin of life. Then some of the definitions of life presently used are cited and briefly discussed, starting with the definition adopted by NASA as a general working definition. It is shown that this is too limited if one wishes to provide a broad encompassing definition, and some extensions of it are presented and discussed. Finally it is shown how the different definitions of life reflect the main schools of thought that presently dominate the field on the origin of life.

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.

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.

Human skin irritation studies of a lecithin microemulsion gel and of lecithin liposomes.

Soybean lecithin microemulsion gels offer promising features for the possible use as matrices in transdermal therapeutic systems. In order to assess the skin irritancy potential of the gel, acute and cumulative irriation tests were performed in human subjects in vivo using as comparison an unilamellar soybean lecithin liposome preparation and the solvent isopropyl palmitate (IPP). Acute irritation was tested in 151 volunteers in a 48-hour patch test, whereas cumulative irritation was assessed in a 21-day human repeated insult patch test in 20 volunteers. In the acute irritation test, discrete irritation (erythema only) developed with the gel in 2 subjects (1.3%), with the liposomes in 3 subjects (2.0%), and with IPP in 2 subjects (1.3%). For the assessment of cumulative irritation, the IT50 (irritation time of 50% of the test population) was calculated. IT50 was 13 days for the gel, 14 days for the liposomes and 17 days for IPP. This study shows a very low acute and a low cumulative irritancy potential for the soybean lecithin microemulsion gel making it a candidate matrix for transdermal therapeutic systems also under toxicological aspects.

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.

Photochemistry of a photosynthetic reaction center immobilized in lipidic cubic phases.

Photosynthetic reaction centers, isolated and purified from the facultative phototrophic bacterium Chloroflexus aurantiacus, were immobilized in optically transparent lipidic cubic phases composed of 42% (w/w) 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine and 58% (w/w) water. The immobilized photosynthetic protein retains its native properties, as indicated by visible and circular dichroic spectra. The ground state visible spectrum of the immobilized reaction centers is very similar to the corresponding spectrum in aqueous solution, indicating that the protein pigments are not extracted into the lipidic regions of the cubic phase. The secondary structure of the protein is maintained in the immobilized state, as determined by far-UV circular dichroism spectroscopy in the 200- to 250-nm range. Moreover, immobilized reaction centers retain their photochemical activity: a reversible photo-oxidation of the primary electron donor (P) is seen upon continuous illumination. Furthermore, the entrappment of reaction centers does not affect the kinetics of charge recombination between the photo-oxidized primary donor (P(+)) and the photoreduced primary quinone acceptor, generated by a short flash of light. Reaction centers devoided of the secondary quinone acceptor can be easily reconstituted in cubic phases by means of their coimmobilization with 1,4-naphtoquinone. Indeed, the kinetics for charge recombination in reconstituted reaction centers is dramatically slower than the corresponding kinetics in the unreconstituted protein. Interestingly, immobilized reaction centers are significantly stabilized as compared with reaction centers in aqueous solution: the integrity of the protein in the cubic phase is maintained for at least 5 months, whereas in water solution 50% of the activity is lost within 2 months. (c) 1995 John Wiley & Sons, Inc.

Enzymatic RNA replication in self-reproducing vesicles: an approach to a minimal cell.

The replication of a RNA template catalyzed by Q beta replicase was obtained in oleic acid/oleate vesicles simultaneously with the self-reproduction of the vesicles themselves. This was accomplished by entrapping the enzyme Q beta replicase, the RNA template, and the ribonucleotides ATP, CTP, GTP, and UTP inside the vesicles. The water-insoluble oleic anhydride was then added externally. It binds to the vesicle bilayer where it is catalytically hydrolyzed yielding the carboxylate surfactant in situ, which then brings about growth and reproduction of the vesicles themselves. This experiment is presented as a first approach to a synthetic minimal cell, in which the reproduction of the membrane and the replication of the internalized RNA molecules proceed simultaneously.

sn-1,2-diacylglycerol cholinephosphotransferase from pig liver: mixed micellar assay and kinetic analysis of the partially pure enzyme.

sn-1,2-Diacylglycerol cholinephosphotransferase from pig liver microsomes was partially purified through a procedure involving solubilization with sodium cholate and chromatography on Sepharose 6B. The resulting preparation was 19-fold enriched with respect to microsomes and was shown to be very sensitive to different detergents. Sodium cholate gave the best yields in activity. In a mixed micellar assay with Triton X-100 a strong dependence of the enzyme activity on the concentration of mixed micelles was observed, due to Triton X-100 acting as an inactivator. Soja phosphatidylcholine added exogenously protected the enzyme against detergent inactivation and stimulated the enzyme activity. Dioleoyl-phosphatidylcholine had a similar stimulatory effect, whereas didecanoyl- or dioctanoyl-phosphatidylcholine did not; thus long-chain phosphatidylcholines seem to be essential in the activation of cholinephosphotransferase. In a mixed micellar assay with sodium cholate no inactivation of the enzyme could be detected and it was found that soja phosphatidylcholine stimulates the activity in a greater extent than in Triton X-100 mixed micelles. The phospholipid activates the enzyme in a noncompetitive way with an activation constant of 176 mol%. Km was estimated as 1.54 mol% with a Vmax = 30 nmol/min/mg protein. Those results support an activation mechanism by phosphatidylcholine interacting at sites different from the active center. The high activation constant led to the conclusion that cholinephosphotransferase requires a lipidic boundary for full activation. No activation by substrate was observed. Short-chain diacylglycerides such as dihexanoyl-, dioctanoyl-, or didecanoylglycerol can be used as substrates although the enzyme in this case has only 5 to 10% of the activity it has for dioleoylglycerol or egg diglycerides.

Surfactant-Induced hydrogen production in cyanobacteria.

Addition of Tween 85 to aqueous suspensions of Anabaena variabilis induced photosynthetic evolution of hydrogen over a time span of several weeks: As much as 148 nmol H(2)/h . mg dry weight was produced in the first week by a suspension containing 4.2 mg dry weight of cells and 77 mM Tween 85. The chemical structure of Tween 85 was a necessary prerequisite for inducing hydrogen production, as compounds such as Tween 20, 60, and 80 had a quite different effect. There was a coupling between photosynthetic oxygen evolution and hydrogen evolution: Hydrogen evolution started to be effective only when oxygen evolution subdued. The presence of heterocysts in A. variabilis was also required for the Tween-induced hydrogen production. Based on these observations, possible mechanisms for the photosynthetic effect of Tween 85 are advanced and discussed.

Spectroscopic and kinetic studies of lipases solubilized in reverse micelles.

The conformation and activity of three different lipases have been studied in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. In the case of human pancreatic lipase, the conformation of the polypeptide chain--as judged from far-UV circular dichroism measurements--is only slightly altered after the enzyme is transferred from a bulk aqueous solution into the microenvironment of reverse micelles. Significant spectral changes in the near-UV circular dichroism and fluorescence spectrum indicate, however, that the solvation of aromatic amino acid side chains is considerably different in reverse micelles. Conversely, the circular dichroism spectra of the lipases from Candida rugosa and Pseudomonas sp. are considerably different in reverse micelles, compared with the spectra in aqueous solution, indicating that both enzymes loose the native structure at the water/AOT/oil interface. Bound substrate and/or product can prevent this denaturation. While Pseudomonas sp. and human pancreatic lipase are inhibited by tetrahydrolipstatin (THL), the lipase from Candida rugosa is not. These data, together with additional activity and inhibition data, indicate that the micellar microenvironment accentuates the difference between the different enzymes in terms of the relation structure/activity.

Reverse micelles in protein separation: the use of silica for the back-transfer process.

In order to use reverse micellar solutions successfully for the separation of proteins, good methods are needed to recover the biomolecules into an aqueous environment after solubilization into organic micellar media. Usually the recovery is accomplished by equilibrating the protein-loaded reverse micellar solution with a water phase containing an appropriate salt (back-transfer). In this article we describe an alternative "back extraction" procedure which is based on the addition of silica to the protein-containing reverse micellar solution. In this way, the water is stripped from the reverse micellar solution. [i.e., bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane/water] and the proteins adsorb to the silica particles. The adsorption process is shown to be practically quantitative. The subsequent recovery of the proteins form the silica into an aqueous solution turns out to be most efficient at alkaline pH (pH 8); 60-80 of the total protein (alpha-chymotrypsin or trypsin) could be recovered. The specific enzyme activity at the end of the whole cycle can be as high as 80-100%. The procedure is applied also for the back extraction from micellar solutions in which, instead of AOT, a biocompatible surfactant such as a synthetic short-chain lecithin was used. It is shown that the recovery of a alpha-chymotrypsin and trypsin is also achievable under these conditions in quite good yield and under good maintenance of the enzyme's catalytic activity.