Cationic phosphoramidate alpha-oligonucleotides efficiently target single-stranded DNA and RNA and inhibit hepatitis C virus IRES-mediated translation

A potential means to improve the efficacy of steric-blocking antisense oligonucleotides (ON) is to increase their affinity for a target RNA. The grafting of cationic amino groups to the backbone of the ON is one way to achieve this, as it reduces the electrostatic repulsion between the ON and its target. We have examined the duplex stabilising effects of introducing cationic phosphoramidate internucleoside linkages into ON with a non-natural α-anomeric configuration. Cationic α-ON bound with high affinity to single-stranded DNA and RNA targets. Duplex stabilisation was proportional to the number of cationic modifications, with fully cationic ON having particularly high thermal stability. The average stabilisation was greatly increased at low ionic strength. The duplex formed between cationic α-ON and their RNA targets were not substrates for RNase H. The penalty in T_m inflicted by a single mismatch, however, was high; suggesting that they are well suited as sequence-specific, steric-blocking, antisense agents. Using a well-described target sequence in the internal ribosome entry site of the human hepatitis C virus, we have confirmed this potential in a cell-free translation assay as well as in a whole cell assay. Interestingly, no vectorisation was necessary for the cationic α-ON in cell culture.     

Oxidative stress in Lactococcus lactis

Lactococcus lactis, the most extensively characterized lactic acid bacterium, is a mesophilic- and microaerophilic-fermenting microorganism widely used for the production of fermented food products. During industrial processes, L. lactis is often exposed to multiple environmental stresses (low and high temperature, low pH, high osmotic pressure, nutrient starvation and oxidation) that can cause loss or reduction of bacterial viability, reproducibility, as well as organoleptic and/or fermentative qualities. Among these stress factors, oxidation can be considered one of the most deleterious to the cell, causing cellular damage at both molecular and metabolic levels. During the last two decades, considerable efforts have been made to improve our knowledge of oxidative stress in L. lactis. Many genes involved with both oxidative stress resistance and control mechanisms have been identified; functionally they seem to overlap. The finding of new genes, and a better understanding of the molecular mechanisms of stress resistance in L. lactis and other lactic acid bacterium, will lead to the construction and isolation of stress-resistant strains. Such strains could be exploited for both traditional and probiotic uses.     

Rescue of intracellularly trapped lutropin receptor exodomain by endodomain and reconstitution of a functional membrane receptor: interaction between exo- and endodomains

The lutropin receptor consists of an extracellular N-terminal half and a membrane-associated C-terminal half. hCG initially binds the exodomain with a high affinity and the resulting complex is thought to interact with the endodomain through a secondary contact generating a hormonal signal. Therefore, the exodomain and endodomain are likely to associate directly or indirectly with each other, but lack of fruitful materials and technology has hampered knowledge about their physical relationship and contact sites. In this work, we engineered a double-recombinant (separate exodomain and endodomain) baculovirus system successfully expressing on the surface of insect cells high levels of split LH receptor, binding the hormone with high affinity and inducing cAMP synthesis. In contrast, the exodomain and endodomain expressed separately were mostly trapped in cells. Our data indicate that the exodomain and endodomain are disulfide linked in the split receptor. When the disulfide links were reduced, the split receptor still induced cAMP up to 60%, which raises the intriguing possibility of a residual induction activity of the endodomain in the absence of high-affinity ligand binding. Our results also underscore that the targeting and transport of the LH receptor to plasma membrane require both domains, whereas each domain is independently sufficient for folding. The expression level of functional lutropin receptors is the highest ever reported. Our system may also be useful for future studies requiring a high amount of soluble secreted exodomain.     

Respiratory chain defects: what do we know for sure about their consequences in vivo?

The function and the structure of mitochondria have been the subject of intensive research since the discovery of these organelles. Yet, the investigation of patients with mitochondrial disease reveals that we do not understand a large part of the underlying pathogenic processes. This has disastrous consequences in terms of the therapy possibly proposed to the patients and their family. An attempt is made in this short review to question our present ideas on the potential consequences of mitochondrial dysfunctions and to enlighten new observations which might be valuable in the understanding of the physiopathology of these diseases.     

Folding of an abridged beta-lactamase

The effects of C-terminal truncation on the equilibrium folding transitions and folding kinetics of B. licheniformis exo small beta-lactamase (ES-betaL) have been measured. ES-betaL lacking 19 residues (ES-betaL(C)(Delta)(19)) has no enzymic activity. Deletion of the last 14 residues produces ES-betaL(C)(Delta)(14), which is 0.1% active. The enzyme lacking nine residues (ES-betaL(C)(Delta)(9)) is nearly fully active, has native optical and hydrodynamic properties, and is protease resistant, a distinguishing feature of the wild-type enzyme. Although ES-betaL(C)(Delta)(9) folds properly, it does so 4 orders of magnitude slower than ES-betaL, making possible the isolation and characterization of a compact intermediate state (I(P) ES-betaL(C)(Delta)(9)). Based on the analysis of folding rates and equilibrium constants, we propose that equilibrium between I(P) ES-betaL(C)(Delta)(9) and other intermediate slow folding. Residues removed in ES-betaL(C)(Delta)(9) and ES-betaL(C)(Delta)(14) are helical and firmly integrated into the enzyme body through many van der Waals interactions involving residues distant in sequence. The results suggest that the deleted residues play a key role in the folding process and also the existence of a modular organization of the protein matrix, at the subdomain level. The results are compared with other examples of this kind in the folding literature.     

Early left ventricular remodelling following acute coronary accident

Ventricular remodelling following acute coronary syndromes is both complex and multiform. It is due to the response of the myocardium to the different agressions associated with these syndromes, in particular the ischemia and necrosis downstream of the occluded artery. We must not however neglect the role of the remodelling of the lesions resulting from spontaneous reperfusion or provoked by the cells and tissues associated with coronary microcirculation embolisms and the no-reflow phenomenon. Acute post-infarct remodelling is dominated by early ventricular dilatation which largely affects late prognosis, necrosis elimination and its replacement by a fibrotic scar in parallel with a compensatory hypertrophy of the non-infarcted myocardium. The diverse cellular and molecular components of this remodelling are increasingly well-known, allowing us to better explain the beneficial effects of the currently available medications and providing us with new potential therapeutic targets. A grading of this knowledge associated with the identification of new risk factors and early therapeutic interventions should help us to further limit the deleterious aspects of this remodelling in the goal of preventing, or at least delaying, the devolution towards heart failure.