Biomedical subjects

Pier Luigi Luisi

Publications and source records attributed to Pier Luigi Luisi.

At least 19 recordsLinked to original sources

PubMed2006

Investigation of de novo totally random biosequences, Part I: A general method for in vitro selection of folded domains from a random polypeptide library displayed on phage.

This paper reports the initial phase of a research aimed at investigating the folding frequency within a large library of polypeptides generated with a totally random sequence by phage-display technique. Resistance to proteolytic digestion has been used as a first, rudimentary folding criterion. The present paper describes, in particular, the development of a phage-display vector which has a selectable N-terminal affinity tag so that, after controlled proteolysis, the tag is cleaved from the phage. This enables the positive selection of phages that carry proteolytically resistant proteins. To test this system, avian pancreatic polypeptide (APP), one of the smallest proteins with a known structure, was chosen as a model, and its gene was inserted in a plasmid that was then used for phage display. A sequence of three amino acids, corresponding to a substrate for thrombin, was introduced at different locations within the APP sequence without significantly modifying the tertiary structure, as determined by circular dichroism (CD) analysis. These sequences were then used to show that the target tripeptide sequence was protected against proteolysis by the overall folding of the chain. Thus, these results show that the method permits the discrimination between folded and unfolded protein domains displayed on phage. The application of this protocol to a large library of totally random polypeptide chains is discussed as a preliminary to successive work, dealing with the production of totally random polypeptide sequences.

Amino Acid Sequence↗

PubMed2006

Investigation of de novo totally random biosequences, Part II: On the folding frequency in a totally random library of de novo proteins obtained by phage display.

We present an investigation on theoretically possible protein structures which have not been selected by evolution and are, therefore, not present on our Earth ('Never Born Proteins' (NBP)). In particular, we attempt to assess whether and to what extent such polypeptides might be folded, thus acquiring a globular protein status. A library (ca. 10(9) clones) of totally random polypeptides, with a length of 50 amino acids, has been produced by phage display. The only structural bias in these sequences is a tripeptide substrate for thrombin: PRG, chosen according to the criteria described in the preceding Part I of this series. The presence of this substrate in an otherwise totally random sequence forms the basis for a qualitative experimental criterion which distinguishes unfolded from folded proteins, as folded proteins are more protected from protease digestion than unfolded ones. The investigation of 79 sequences, randomly selected from the initially large library, shows that over 20% of this population is thrombin-resistant, likely due to folding. Analysis of the amino acid sequences of these clones shows no significant homology to extant proteins, which indicates that they are indeed totally de novo. A few of these sequences have been expressed, and here we describe the structural properties of two thrombin-resistant randomly selected ones. These two de novo proteins have been characterized by spectroscopic methods and, in particular, by circular dichroism. The data show a stable three-dimensional folding, which is temperature-resistant and can be reversibly denatured by urea. The consequences of this finding within a library of 'Never Born Proteins' are discussed in terms of molecular evolution.

Circular Dichroism↗

PubMed2006

Investigation of de novo totally random biosequences, Part III: RNA Foster: A novel assay to investigate RNA folding structural properties.

Fold is essential to RNA properties, and, in particular, its thermodynamic stability can be used to monitor RNA-protein or RNA-ligand interactions, and to engineer RNA with novel or improved properties. While clearly valuable, experimental determination of RNA folding stability by traditional biophysical techniques requires substantial amounts of pure sample and rather expensive equipment. In this paper, we report a new, simple approach to the determination of RNA folding stability by coupling enzymatic digestion and temperature denaturation. The assay, named RNA folding stability Test (RNA Foster), is designed to probe the fraction of folded RNA (f(fold)) in an equilibrium mixture of folded and unfolded ones as a function of temperature. The simplicity of RNA Foster suggests that it can easily be scaled up for high-throughput studies of RNA folding stability both in basic and applied research.

Base Sequence↗

PubMed2006

Investigation of de novo totally random biosequences, Part IV: Folding Properties of de novo, totally random RNAs.

This work lies within the framework of a broader project aimed at exploring the realm of all possible folded polypeptides; the main question addressed here is whether the corresponding RNAs also assume a folded conformation. We present an investigation on the structural properties of de novo, totally random RNAs by means of the 'RNA Foster' assay. Experimental results show that all RNAs studied are folded at 37 degrees , so that fold seems to be a common feature of RNAs. Random RNAs' fold shows a surprising thermal stability with an average T(m) value at ca. 50 degrees which prompts the idea that thermo-stable structures might not be as rare as they are commonly thought to be. The results are discussed within the general framework of random RNA properties such as those that might have been produced in a prebiotic scenario.

Base Composition↗

PubMed2006

The production of de novo folded proteins by a stepwise chain elongation: a model for prebiotic chemical evolution of macromolecular sequences.

We describe an experimental procedure to mimic the formation of long (over 40 residues) co-oligopetide sequences in many identical copies which may have occurred in the prebiotic molecular evolution. The basic hypothesis is that chain formation is based on the stepwise fragment condensation of randomly generated short oligopeptides, whereby the elongation takes place under the contingent environmental constraints (solubility, pH, salinity), which eliminate most of the products, and thus determine the selection towards one particular small set of chains. The present work aims at verifying the validity of this scheme. In order to do so, we utilize a classic synthetic procedure based on the Merrifield solid-phase synthesis of peptides for the synthesis of randomly produced peptides as well as for their stepwise fragment condensation. Thus, starting from a library of peptides with n=10, the first condensation step produces a library of 16 peptides with 20 residues each (n=20), of which only four remain water-soluble and, therefore, capable to undergo the next fragment condensation step. This gives rise to 16 peptides with n=30, out of which twelve precipitate out under the chosen pH and buffer conditions and are eliminated. Finally, a 44-residue-long water-soluble de novo protein is obtained. This has no homologies or similarities with extant proteins, and, based on circular dichroism (CD), it assumes a stable three-dimensional folding. In agreement with CD data, molecular-modelling simulations suggest an helical fold for the protein with poor, if any, structural homology with known proteins. The implication of this procedure as a general mechanism for the etiology of de novo macromolecular sequences and globular proteins in the origin of life is briefly discussed.

Amino Acid Sequence↗

PubMed2006

Approaches to semi-synthetic minimal cells: a review.

Following is a synthetic review on the minimal living cell, defined as an artificial or a semi-artificial cell having the minimal and sufficient number of components to be considered alive. We describe concepts and experiments based on these constructions, and we point out that an operational definition of minimal cell does not define a single species, but rather a broad family of interrelated cell-like structures. The relevance of these researches, considering that the minimal cell should also correspond to the early simple cell in the origin of life and early evolution, is also explained. In addition, we present detailed data in relation to minimal genome, with observations cited by several authors who agree on setting the theoretical full-fledged minimal genome to a figure between 200 and 300 genes. However, further theoretical assumptions may significantly reduce this number (i.e. by eliminating ribosomal proteins and by limiting DNA and RNA polymerases to only a few, less specific molecular species). Generally, the experimental approach to minimal cells consists in utilizing liposomes as cell models and in filling them with genes/enzymes corresponding to minimal cellular functions. To date, a few research groups have successfully induced the expression of single proteins, such as the green fluorescence protein, inside liposomes. Here, different approaches are described and compared. Present constructs are still rather far from the minimal cell, and experimental as well as theoretical difficulties opposing further reduction of complexity are discussed. While most of these minimal cell constructions may represent relatively poor imitations of a modern full-fledged cell, further studies will begin precisely from these constructs. In conclusion, we give a brief outline of the next possible steps on the road map to the minimal cell.

Animals↗

PubMed2006

From Never Born Proteins to Minimal Living Cells: two projects in synthetic biology.

The Never Born Proteins (NBPs) and the Minimal Cell projects are two currently developed research lines belonging to the field of synthetic biology. The first deals with the investigation of structural and functional properties of de novo proteins with random sequences, selected and isolated using phage display methods. The minimal cell is the simplest cellular construct which displays living properties, such as self-maintenance, self-reproduction and evolvability. The semi-synthetic approach to minimal cells involves the use of extant genes and proteins in order to build a supramolecular construct based on lipid vesicles. Results and outlooks on these two research lines are shortly discussed, mainly focusing on their relevance to the origin of life studies.

Cell Membrane↗

PubMed2005

Condensed DNA in lipid microcompartments.

DNA was studied in lipid reverse micelles with the aim of investigating the interactions of DNA with lipids in a restricted compartment with minimal water content. Circular dichroic (CD) spectra of DNA at low water content showed the characteristic polymer-salt-induced (psi) spectra of condensed DNA. Dynamic light scattering showed a peak around a radius of 400 nm (corresponding to DNA-containing micelles), and a peak around 2.5 nm (corresponding to "empty" micelles). Fourier Transform-IR (FT-IR) spectroscopy was carried out and analyzed in terms of three distinct states of water inside the micelle water pool, where the local concentration of DNA reached an estimated value of ca. 600 mg/mL, comparable to that found in restricted biological compartments.

Animals↗

PubMed2005

RNA selectively interacts with vesicles depending on their size.

RNA and vesicles are two important molecular classes in the origin of life and early evolution, but they are not generally considered as interacting partners. The present paper reports about the interaction between tRNA (Esherichia coli) and vesicles made of the zwitterionic surfactant POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), partially positively charged with small molar fractions (max 10%) of the single-chained CTAB (cetyltrimethylammonium bromide). CTAB is capable to insert efficiently in POPC vesicles (as determined by zeta-potential measurements), and the binding of tRNA to such charged vesicles operates a strong selection being critically dependent upon the vesicle size. The binding of tRNA to the vesicles is size-selective as it induces a strongly pronounced process of aggregation of large vesicles (ca. 160-nm diameter) but not of small ones (ca. 80-nm diameter) that are stable against vesicle aggregation (as followed by dynamic light-scattering and optical density measurements). The aggregation of the large vesicles is fully reversible upon the addition of RNase A. The selective behavior of tRNA with respect to differently sized vesicles is observable in separated samples as well as in a mixture of both populations. In the latter case, the fraction of large vesicles readily aggregates in the presence of the small ones that remain unaltered in the mixture. This kind of discrimination capability of RNA might have been of importance in the early phases of the formation of the protocells.

Cetrimonium↗

PubMed2005

Effect of tryptophan oligopeptides on the size distribution of POPC liposomes: a dynamic light scattering and turbidimetric study.

A chemical regulation of POPC liposome size distribution was investigated, based on the affinity of indole-containing compounds for phosphocholine membranes. In particular, tryptophan oligopeptides have shown interesting properties of size regulation, both when liposomes were formed in their presence and when the peptides were added to a preformed liposome suspension. Combining dynamic light scattering (DLS) and turbidimetric data, it was possible to show how such peptides had an influence on the size distribution of spontaneously formed liposomes prepared by the thin film hydration, reverse-phase evaporation and ethanol (or methanol) injection methods. In the presence of Trp-Trp or Trp-Trp-Trp, a disappearance of large vesicle aggregates was observed, as suggested also by light microscopy analysis. On the contrary, no effect was detected using extruded vesicles. Turbidimetric titration allowed the determination of the relative efficacy of the size regulators, Trp-Trp-Trp being about 20 times more powerful than the dimer, while the monomer had no effect. In addition, other indole-containing compounds and the antimicrobial peptide indolicidin were tested, showing similar behaviours. Discussing the results according to the current knowledge about the preference of Trp residues for interfacial regions in lecithin bilayers, this study confirms the relevant role of tryptophan in the biomembrane binding properties of many peptides and introduces a new behavior in the field of liposomes-peptides interactions.

Indoles↗

PubMed2004

Cell-free protein synthesis through solubilisate exchange in water/oil emulsion compartments.

This work is aimed at finding conditions under which synthetic compartments used as cell models can fuse with each other and allow reagents contained in the different compartments to react. This goal seems to be best achieved by the use of water in oil emulsions (w/o) with dimensions in the range of 30-60 microm. In particular, cell-free EGFP (enhanced green fluorescent protein) synthesis takes place in Tween 80/Span 80 w/o emulsions, and the extent of the reaction can be monitored directly by fluorescence. The medium is mineral oil, containing 0.5 % v/v aqueous solution. Different premixing configurations of the components (plasmid, amino acids, E. Coli extract) are used and compared. The in vitro synthesis of EGFP in emulsion droplets proceeds for 1 h, and the yield is 7.5 ng microL(-1) protein. EGFP synthesis in aqueous solution takes place for at least 5 h. The yield is 10.5 ng microL(-1) protein after 1 h and 15.8 ng microL(-1) protein after 5 h. The results with the w/o emulsions show that solubilisate exchange takes place among the different water droplets, but it is not possible to demonstrate clearly that a true fusion takes place.

Cell-Free System↗

PubMed2004

Matrix effect in oleate micelles-vesicles transformation.

It is accepted by many authors that the formation of closed molecular structures is a key step in the evolution of life. Oleate vesicles represent a good model system in this framework due to the fact that they self-assemble spontaneously and that fatty acids are considered as possible prebiotic structures. In this contribution, we will focus the attention on the transition from oleate micelles to oleic acid/oleate vesicles induced by a pH change. This transformation is strongly influenced by the presence of pre-formed vesicles. We called this phenomenon the matrix effect. The influence of pre-added POPC liposomes (POPC = 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine) and oleic acid/oleate vesicles on the process rate and on the final size distribution will be discussed elucidating the main differences between these two systems.

Micelles↗

PubMed2004

Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection method.

Small-sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug-containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) then on the novel L-carnitine derivative PUCE (palmitoyl-(R)-carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of "multiple injection method" was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan-containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI-H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no-liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water-insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.

Animals↗

PubMed2004

A possible route to prebiotic vesicle reproduction.

Spherical bounded structures such as those formed by surfactant aggregates (mostly micelles and vesicles), with an inside that is chemically and physically different from the outside medium, can be seen as primitive cell models. As such, they are fundamental structures for the theory of autopoiesis as originally formulated by Varela and Maturana. In particular, since self-reproduction is a very important feature of minimal cellular life, the study of self-reproduction of micelles and vesicles represents a quite challenging bio-mimetic approach. Our laboratory has put much effort in recent years into implementing self-reproduction of vesicles as models for self-reproduction of cellular bounded structures, and this article is a further contribution in this direction. In particular, we deal with the so-called matrix effect of vesicles, related to the fact that when fresh surfactant is added to an aqueous solution containing preformed vesicles of a very narrow size distribution, the newly formed vesicles (instead of being polydisperse, as is usually the case) have dimensions very close to those of the preformed ones. In practice, this corresponds to a mechanism of reproduction of vesicles of the same size. In this article, the matrix effect is re-elaborated in the perspective of the origin of life, and in particular in terms of the prebiotic mechanisms that might permit the growth and reproduction of vesicles. The data are analyzed by dynamic light scattering with a new program that permits the calculation of the number-weighted size distribution. It is shown that, on adding a stoichiometric amount of oleate micelles to preformed oleate vesicles extruded at 50 and 100 nm, the final distribution contains about twice the initial number of particles, centered around 50 and 100 nm. The same holds when oleate is added to preformed phospholipid liposomes. By contrast, when the same amount of oleate is added to an aqueous solution (as a control experiment), a very broad distribution ranging between 20 and 1000 nm is obtained. The data can then be seen as a kind of reproduction of the same size vesicles, and the argument is advanced that this may correspond to a simple prebiotic mechanism of vesicle multiplication in prebiotic times, when only physical forces might be responsible for the basic mechanisms of early protocell growth and division. Preliminary data also show that repeated addition of oleate maintains the same basic initial features, and that surfactants other than oleate also respect the reproductive mode of the matrix effect.

Cell Physiological Phenomena↗

PubMed2003

Contingency and determinism.

Contingency versus determinism is an old, classic issue in the history of science, and certain trends of contemporary science literature appear to be bringing it to the foreground again.

Biological Evolution↗

PubMed2003

Autopoiesis: a review and a reappraisal.

The aim of the paper is to review critically the notion of autopoiesis as presented by Maturana and Varela. In particular, recognizing that there are difficulties in obtaining a complete and clear picture from the primary literature, an effort is made to present a coherent view-also based on many years of personal contact with Francisco Varela. The paper begins with a few historical notes to highlight the cultural background from which the notion of autopoiesis arose. The basic principles of autopoiesis as a theory of cellular life are then described, emphasizing also what autopoiesis is not: not an abstract theory, not a concept of artificial life, not a theory about the origin of life-but rather a pragmatic blueprint of life based on cellular life. It shown how this view leads to a conceptually clear definition of minimal life and to a logical link with related notions, such as self-organization, emergence, biological autonomy, auto-referentiality, and interactions with the environment. The perturbations brought about by the environment are seen as changes selected and triggered by the inner organization of the living. These selective coupling interactions impart meaning to the minimal life and are thus defined by Maturana and Varela with the arguable term of "cognition". This particular view on the mutual interactions between living organism and environment leads these authors to the notion of "enaction", and to the surprising view that autopoiesis and cognition are two complementary, and in a way equivalent, aspects of life. It is then shown how cognition, so defined, permits us to build a bridge between biology and cognitive science. Autopoiesis also allows one to conceive chemical models of minimal cellular life that can be implemented experimentally. The corresponding work on "chemical autopoiesis" is then reviewed. The surprising impact of autopoiesis in the social sciences ("social autopoiesis") is also briefly discussed. This review also comments on why the theory of autopoiesis had, and still has, a difficult time being accepted into the mainstream of life-science research. Finally, it is pointed out that the new interest in system biology and complexity theories may lead to a reappraisal of autopoiesis and related notions, as outlined also by other authors, such as Tibor Ganti and Stuart Kauffmann.

Cell Cycle↗

PubMed2002

Toward the engineering of minimal living cells.

The article focuses on the notion of a synthetic or semi-synthetic minimal cell, defined as a system that has the minimal and sufficient structural conditions for cellular life. It is emphasized that two complementary approaches are in principle possible, defined as "bottom-up" and "top-down" approaches. The first one aims at the construction of a minimal cell starting from scratch, and it is argued that a very serious bottle-neck to this pathway lies in the origination of specific macro-molecular sequences, as in nature those were constructed most likely by a particular contingent set of conditions. The top-down approaches utilize extant genes and enzymes, and the work in this case is based on the incorporation of the minimal and sufficient amount of such macromolecules into liposomes, as models for the shell of biological cells. The first phase of this ambitious project foresees the study of conditions under which complex molecular biology reactions takes place in the compartments of liposomes. Examples of these reactions are provided, for example, the production of RNA throughout Q-beta replicase in a self-reproducing vesicle system; or PC Reaction in phospholipid vesicles; or even the incorporation of ribosomes in liposomes, with the production of polypeptide chains. The use of giant vesicles is also illustrated. These systems, due to their large size, offer the advantage that by way of special micro-injection techniques, all sort of biochemical agents can be directly introduced in the compartment; and that the reaction can be followed by optical microscopy. In the final part of the article, the outlook of increasing the complexity of these liposome systems so as to arrive at first semi-synthetic cells is discussed.

Biopolymers↗

PubMed2002

Circular dichroic properties and average dimensions of DNA-containing reverse micellar aggregates.

With the aim of investigating the compartmentation of nucleic acids and surfactant aggregates, we have studied the circular dichroic properties of DNA solubilized in reverse micelles. DNA incorporated in AOT/isooctane reverse micelles (AOT=bis-2-ethyl-hexyl sodium sulfosuccinate) assumes an anomalous circular dichroism (CD) spectrum with the characteristic features of a psi spectrum. Older literature observations could therefore be confirmed that attribute these spectral changes to the fact that the reverse micelles induce the formation of a condensed form of DNA. A dynamic light scattering (DLS) characterization of the DNA-containing micellar solutions was carried out, and three populations of aggregates in a polar solvent are observed, with an average radius centered at 5, 100 and 1000 nm, respectively, all three containing DNA. Several forms of DNA, including a plasmid, have been investigated. The formation of 1 microm-large aggregates depends on the DNA concentration and such aggregates disappear in the course of a few hours. Conversely, the 100 nm aggregates are stable for at least 1 day and contain DNA in a normal spectral state at low concentration and in a condensed form-it is the characteristic psi spectrum-in a higher concentration range. The solubilization of DNA in reverse micelles brings about unexpected larger structures in hydrocarbon solution, and whereas the very large component can be with all likelihood be attributed to clusters of smaller reverse micelles, the components at 100 nm radius appear to be a quite stable and characteristic feature of DNA-containing reverse micelles.

Animals↗