The origin of kinetic cooperativity in prehiotic catalysts

A polyallylamine carrying long hydrophobic dodecyl groups and adenine residues as side chains (PALAD C₁₂) may be able to catalyze the hydrolysis ofN-carbobenzoxy-l-alaninep-nitrophenyl ester (N-Cbz-Ala) as well asp-nitrophenyl acetate (pNPA). The progress curve of hydrolysis of the former displays a long lag and apparently no steady state. After this transient the rate falls off due to the accumulation of the products. Conversely, the hydrolysis ofp-nitrophenyl acetate displays classical burst kinetics followed by a slow decline of the reaction rate. Theoretical considerations show that a steady state may be expected to occur only if the concentration of the free catalyst is very small during the reaction. This condition is sufficient to allow the rate of disappearance of the substrate to be equal to the rate of appearance of the products, which is precisely a condition for the existence of a steady state. If the catalyst is poorly active and has a loose affinity for its substrate and product, the measurement of a significant reaction rate will require a much larger concentration of the catalyst. Therefore, under these conditions, one cannot expect a steady state to occur. The mathematical expression of the error made in the steady-state assumption has been derived. This error increases with the catalyst concentration and decreases if the affinity of the substrate for the catalyst is high. Therefore the lack of steady state is associated with the affinity (or the dissociation) of the substrate and the product for the catalyst. When this affinity is low, the free concentration of the catalyst during the reaction is high and one cannot expect a steady state to occur. This is precisely what takes place with N-Cbz-Ala. A mathematical expression of the rate of hydrolysis of N-Cbz-Ala and of any reactant that displays this type of kinetics may be derived at the end of the transient when the rate is close to its maximum value. Under these conditions the rate cannot follow classical Michaelis-Menten kinetics and displays positive cooperativity. It may therefore be speculated that primordial template-like catalysts that were displaying a poor affinity for their substrates and products were already exhibiting apparent positive cooperativity in the kinetic reactions they were able to catalyze. Correspondence to: J. Ricard     

Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT)

Lecithin cholesterol acyltransferase (LCAT) is an interfacial enzyme active on both high-density (HDL) and low-density lipoproteins (LDL). Threading alignments of LCAT with lipases suggest that residues 50-74 form an interfacial recognition site and this hypothesis was tested by site-directed mutagenesis. The (delta56-68) deletion mutant had no activity on any substrate. Substitution of W61 with F, Y, L or G suggested that an aromatic residue is required for full enzymatic activity. The activity of the W61F and W61Y mutants was retained on HDL but decreased on LDL, possibly owing to impaired accessibility to the LDL lipid substrate. The decreased activity of the single R52A and K53A mutants on HDL and LDL and the severer effect of the double mutation suggested that these conserved residues contribute to the folding of the LCAT lid. The membrane-destabilizing properties of the LCAT 56-68 helical segment were demonstrated using the corresponding synthetic peptide. An M65N-N66M substitution decreased both the fusogenic properties of the peptide and the activity of the mutant enzyme on all substrates. These results suggest that the putative interfacial recognition domain of LCAT plays an important role in regulating the interaction of the enzyme with its organized lipoprotein substrates.     

Role of temperature as a triggering signal for organogenesis or somatic embryogenesis in wounded leaves of chicory cultured in vitro

Static liquid half-strength Murashige and Skoog medium, containing10.1 mM KCI instead of KNO3, 1.7 mM glutamine, microelements,vitamins, 60 mM sucrose, 0.1 //M a-naphthaleneacetic acid and2.5//M 2-isopentenyladenine allows either somatic embryo-genesisor shoot organogenesis along the borders of leaf incisions ofa Cichorium hybrid [Cichorium intybus L. x Cichorium endiviaL.). These phenomena are temperature-dependent and the latterpromotes the development of callus and shoots at 20 °C and25 °C instead of direct somatic embryogenesis at 35 °C.At 30 °C all types of morphogenesis are observed. After5 d of culture, cells grown at each temperature exhibit enlargednuclei with prominent nucleoli, fragmented vacuoles and densecytoplasm. However, at 25 °C, callose is restricted to woundedcells whereas at 35 °C some nearby mesophyll cells showa more or less complete callose sheath. On the 7th day, proembryosat 35 °C show a superficial network which is absent at 25°C on shoot primordia which are covered with a smooth precociousproto-derm. Why do activated cells undergo segmentation andsomatic embryogenesis at 35 °C instead of ordinary mitosiswith callus and shoot formation at 20 °C and 25 °C? Key words: Cichorium, morphogenesis, somatic embryos, tpmnpratnrp     

Contribution of the hydrophobicity gradient to the secondary structure and activity of fusogenic peptides

Fusogenic peptides belong to a class of helical amphipathic peptides characterized by a hydrophobicity gradient along the long helical axis. According to the prevailing theory regarding the mechanism of action of fusogenic peptides, this hydrophobicity gradient causes the tilted insertion of the peptides in membranes, thus destabilizing the lipid core and, thereby, enhancing membrane fusion. To assess the role of the hydrophobicity gradient upon the fusogenic activity, two of these fusogenic peptides and several variants were synthesized. The LCAT-(57-70) peptide, which is part of the sequence of the lipolytic enzyme lecithin cholesterol acyltransferase, forms stable beta-sheets in lipids, while the apolipoprotein A-II (53-70) peptide remains predominantly helical in membranes. The variant peptides were designed through amino acid permutations, to be either parallel, perpendicular, or to retain an oblique orientation relative to the lipid-water interface. Peptide-induced vesicle fusion was monitored by lipid-mixing experiments, using fluorescent probes, the extent of peptide-lipid association, the conformation of lipid-associated peptides and their orientation in lipids, were studied by Fourier Transformed Infrared Spectroscopy. A comparison of the properties of the wild-type and variant peptides shows that the hydrophobicity gradient, which determines the orientation of helical peptides in lipids and their fusogenic activity, further influences the secondary structure and lipid binding capacity of these peptides.     

8-Azidoadenosine and ribonucleotide reductase.

Inhibitors of ribonucleotide reductase are potential antiproliferative agents, since they deplete cells from DNA precursors. Substrate nucleoside analogues, carrying azido groups at the base moiety, are shown to have strong cytostatic properties, as measured by the inhibition of the incorporation of thymidine into DNA. One compound, 8-azidoadenosine, inhibits CDP reduction in cytosolic extracts from cancer cells. The corresponding diphosphate behaves as a substrate for ribonucleotide reductase while the triphosphate is an allosteric effector.     

The Pseudomonas aeruginosa membranes: a target for a new amphiphilic aminoglycoside derivative?

Aminoglycosides are among the most potent antimicrobials to eradicate Pseudomonas aeruginosa. However, the emergence of resistance has clearly led to a shortage of treatment options, especially for critically ill patients. In the search for new antibiotics, we have synthesized derivatives of the small aminoglycoside, neamine. The amphiphilic aminoglycoside 3',4',6-tri-2-naphtylmethylene neamine (3',4',6-tri-2NM neamine) has appeared to be active against sensitive and resistant P. aeruginosa strains as well as Staphylococcus aureus strains (Baussanne et al., 2010). To understand the molecular mechanism involved, we determined the ability of 3',4',6-tri-2NM neamine to alter the protein synthesis and to interact with the bacterial membranes of P. aeruginosa or models mimicking these membranes. Using atomic force microscopy, we observed a decrease of P. aeruginosa cell thickness. In models of bacterial lipid membranes, we showed a lipid membrane permeabilization in agreement with the deep insertion of 3',4',6-tri-2NM neamine within lipid bilayer as predicted by modeling. This new amphiphilic aminoglycoside bound to lipopolysaccharides and induced P. aeruginosa membrane depolarization. All these effects were compared to those obtained with neamine, the disubstituted neamine derivative (3',6-di-2NM neamine), conventional aminoglycosides (neomycin B and gentamicin) as well as to compounds acting on lipid bilayers like colistin and chlorhexidine. All together, the data showed that naphthylmethyl neamine derivatives target the membrane of P. aeruginosa. This should offer promising prospects in the search for new antibacterials against drug- or biocide-resistant strains.     

Sequence-targeted photosensitized reactions in nucleic acids by oligo-alpha-deoxynucleotides and oligo-beta-deoxynucleotides covalently linked to proflavin

Proflavin was covalently linked to the 3'-end or to the 5'-end of an octadeoxythymidylate. This oligonucleotide was synthesized with either the natural beta-anomer of thymidine or its synthetic alpha-anomer. A polymethylene chain was used to link one of the amino groups of proflavin to a terminal thiophosphate group of the oligonucleotide. A 27-mer oligodeoxynucleotide containing an octadeoxyadenylate sequence was used as a target for the proflavin-substituted octadeoxythymidylates. Upon irradiation with visible light, photo-cross-linking reactions induced the formation of branched species that migrated more slowly than the 27-mer on denaturing polyacrylamide gels. Piperidine treatment of the photo-cross-linked species induced strand breaks in the 27-mer. In addition, proflavin induced photosensitized reactions at guanine residues in the 27-mer sequence which were converted to strand breaks following piperidine treatment. Triple-helix formation by the oligothymidylates with their complementary oligodeoxyadenylate sequence at high salt concentration led to photo-cross-linking and cleavage reactions on both sides of the target sequence. These results show that it is possible to target photosensitized reactions to specific sequences on nucleic acids. This opens new possibilities for site-directed mutagenesis and the development of photoactive anti-messenger oligodeoxynucleotides.     

Oligoribonucleotide Synthesis by the Phosphite Procedure from 2′-Dimethoxytritylated Nucleosides

Abstract

2′-Dimethoxytritylated ribonucleosides were prepared and condensed with cyanoethyl phosphodichloridite to form oligoribo-nucleotides.     

Sequence-specific recognition, photocrosslinking and cleavage of the DNA double helix by an oligo-[alpha]-thymidylate covalently linked to an azidoproflavine derivative.

A 3-azidoproflavine derivative was covalently linked to the 5'-end of an octathymidylate synthesized with the [alpha]-anomers of the nucleoside. Two target nucleic acids were used for this substituted oligo-[alpha]-thymidylate: a 27-mer single-stranded DNA fragment containing an octadeoxyadenylate sequence and a 27-mer duplex containing eight contiguous A.T base pairs with all adenines on the same strand. Upon visible light irradiation the octa-[alpha]-thymidylate was photocrosslinked to the single-stranded 27-mer. Chain breaks were induced at the crosslinked sites upon piperidine treatment. From the location of the cleavage sites on the 27-mer sequence it was concluded that a triple helix was formed by the azidoproflavine-substituted oligo-[alpha]-thymidylate with its complementary oligodeoxyadenylate sequence. When the 27-mer duplex was used as a substrate cleavage sites were observed on both strands after piperidine treatment of the irradiated sample. They were located at well defined positions which indicated that the octathymidylate was bound to the (dA)8.(dT)8 sequence in parallel orientation with respect to the (dA)8-containing strand. Specific binding of the [alpha]-octathymidylate involved local triple strand formation with the duplex (dA)8.(dT)8 sequence. This result shows that it is possible to synthesize sequence-specific molecules which specifically bind oligopurine-oligopyrimidine sequences in double-stranded DNA via recognition of the major groove hydrogen bonding sites of the purines.     

Design of molecules that specifically recognize and cleave apurinic sites in DNA

We have prepared a series of a tailor-made molecules that recognize and cleave DNA at apurinic sites in vitro. These molecules incorporate in their structure different units designed for specific function: an intercalator for DNA binding, an nucleic base for abasic site recognition and a linking chain of variable length and nature (including amino and/or amido functions). The cleavage efficiency of the molecules can be modulated by varying successively the nature of the intercalating agent, the nucleic base and the chain. All molecules bind to native calf thymus DNA with binding constants ranging from 10⁴ to 10⁶ M^?1. Their cleavage activity was determined on plasmid DNA (pBR 322) containing 1.8 AP-sites per DNA-molecule. The minimum requirements for cleavage are the presence of the three units, the intercalator, the nucleic base and at least one amino function in the chain. The most efficient molecules cleaved plasmid DNA at nanomolar concentrations. Enzymatic experiments on the termini generated after cleavage of AP-DNA suggest a strand break induced by a β-elimination reaction. In order to get insight into the mode of action (efficiency, selectivity, interaction), we have used synthetic oligonucleotides containing either a true abasic site at a determined position to analyse the cleavage parameters of the synthetic molecules by HPLC or a chemically stable along (tetrahydrofuran) of the abasic site for high field ¹H NMR spectrometry and footprinting experiments. All results are consistent with a β-elimination mechanism in which each constituent of the molecule exerts a specific function as indicated in the scheme: DNA targeting, abasic site nucleases and can be used advantageously as substitutes for the natural enzyme for in vitro cleavage of AP-sites containing DNA.