Biomedical subjects

P A Monnard

Publications and source records attributed to P A Monnard.

4 recordsLinked to original sources

PubMed2001

Eutectic phases in ice facilitate nonenzymatic nucleic acid synthesis.

Polymeric compounds similar to oligonucleotides are relevant to the origin of life and particularly to the concept of an RNA world. Although short oligomers of RNA can be synthesized nonenzymatically under laboratory conditions by second-order reactions in concentrated solutions, there is no consensus on how these polymers could have been synthesized de novo on the early Earth from dilute solutions of monomers. To address this question in the context of an RNA world, we have explored ice eutectic phases as a reaction medium. When an aqueous solution freezes, the solutes become concentrated in the spaces between the ice crystals. The increased concentration offsets the effect of the lower temperature and accelerates the reaction. Here we show that in the presence of metal ions in dilute solutions, frozen samples of phosphoimidazolide-activated uridine react within days at -18 degrees C to form oligouridylates up to 11 bases long. Product yields typically exceed 90%, and approximately 30% of the oligomers include one or more 3'-5' linkages. These conditions facilitate not only the notoriously difficult oligouridylate synthesis, but also the oligomerization of activated cytidylate, adenylate, and guanylate. To our knowledge, this represents the first report to indicate that ice matrices on the early Earth may have accelerated certain prebiotic polymerization reactions.

Biological Evolution↗

PubMed1999

Enzymatic reactions in liposomes using the detergent-induced liposome loading method.

Microcompartmentalization is a crucial step in the origin of life. More than 30 years ago, Oparin et al. proposed models based on biochemical reactions taking place in so-called coacervates. Their intention was to develop systems with which semipermeable microcompartments could be established. In the present work we follow their intuition, but we use well-characterized bilayer structures instead of the poorly characterized coacervates. Liposomes from phospholipids can be used as microreactors but they exhibit only a modest permeability and, therefore, chemical reactions occurring inside these structures are depleted after a relatively short period. Here it is shown that even highly stable liposomes from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be used as semipermeable microreactors when treated with sodium cholate. Using this kind of mixed liposomes, we describe a biochemical reaction occurring inside the liposomes while the same reaction is prevented in the external medium. In addition, we show that this cholate-induced permeability of POPC bilayers can even be used to load macromolecules such as enzymes from the outside.

Cholates↗

PubMed1997

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.

Adenosine Triphosphate↗

PubMedDate not supplied

Nutrient uptake by protocells: a liposome model system.

Over the past decade, several liposome-based models for protocells have been developed. For example, liposome systems composed of polymerase enzymes encapsulated with their substrates have demonstrated that complex compartmentalized reactions can be carried out under conditions in which polymeric products are protected from degradation by hydrolytic enzymes present in the external medium. However, such systems do not have nutrient uptake mechanisms, which would be essential for primitive cells lacking the highly evolved nutrient transport processes present in all contemporary cells. In this report, we explore passive diffusion of solutes across lipid bilayers as one possible uptake mechanism. We have established conditions under which ionic substrates as large as ATP can permeate bilayers at rates capable of supplying an encapsulated template-dependent RNA polymerase. Furthermore, while allowing the permeation of monomer substrates such as ATP, bilayer vesicles selectively retained polymerization products as small as dimers and as large as a transfer RNA. These observations demonstrate that passive diffusion could be used by the earliest forms of cellular life for transport of important nutrients such as amino acids, phosphate, and phosphorylated organic solutes.

Adenosine Triphosphate↗