Systematic screening of LNA/2'-O-methyl chimeric derivatives of a TAR RNA aptamer

We synthesized and evaluated by surface plasmon resonance 64 LNA/2'-O-methyl sequences corresponding to all possible combinations of such residues in a kissing aptamer loop complementary to the 6-nt loop of the TAR element of HIV-1. Three combinations of LNA/2'-O-methyl nucleoside analogues where one or two LNA units are located on the 3' side of the aptamer loop display an affinity for TAR below 1nM, i.e. one order of magnitude higher than the parent RNA aptamer. One of these combinations inhibits the TAR-dependent luciferase expression in a cell assay.     

Evolution of ancient satellite DNAs in sturgeon genomes

This study characterizes a repetitive DNA family of sequences in sturgeon, the PstI satellite DNA. We have found a high degree of preservation for these sequences, which are present in all 13 species analyzed, including within the genera Acipenser, Huso, and Scaphirhynchus of the family Acipenseridae. This is one of the most ancient satellite DNAs found to date, because it has been estimated to be more than 100 million years old. Alternatively, to the current view that most satellite DNAs are species-specific or preserved in a few closely related species, the PstI family and other previously characterized sturgeon satellite DNA, the HindIII, represent the most fascinating exceptions to the rapid sequence change usually undergone by satellite DNAs. Here, we compare the evolutionary pattern of these two satellite DNA families, PstI and HindIII, which differ markedly in length, sequence, and nucleotide composition. We have found that, in contrast to the situation in most other living beings, a high degree of preservation, a slow sequence change rate and slowed concerted evolution, appears to be a general rule for sturgeon satellite DNAs. The possible causes for all these features are discussed in the light of the evolutionary specifics found within these ancient organisms.     

Co-expression of metabotropic glutamate receptor 7 and N-type Ca(2+) channels in single cerebrocortical nerve terminals of adult rats

The modulation of calcium channels by metabotropic glutamate receptors (mGluRs) is a key event in the fine-tuning of neurotransmitter release. Here we report that, in cerebrocortical nerve terminals of adult rats, the inhibition of glutamate release is mediated by mGluR7. In this preparation, the major component of glutamate release is supported by P/Q-type Ca2+ channels (72.7%). However, mGluR7 selectively reduced the release component that is associated with N-type Ca2+ channels (29.9%). Inhibition of P/Q channels by mGluR7 is not masked by the higher efficiency of these channels in driving glutamate release when compared with N-type channels. Thus, activation of mGluR7 failed to reduce the release associated with P/Q channels when the extracellular calcium concentration, ([Ca2+]o), was reduced from 1.3 to 0.5 mm. Through Ca2+ imaging, we show that Ca2+ channels are distributed in a heterogeneous manner in individual nerve terminals. Indeed, in this preparation, nerve terminals were observed that contain N-type (31.1%; conotoxin GVIA-sensitive) or P/Q-type (64.3%; agatoxin IVA-sensitive) channels or that were insensitive to these two toxins (4.6%). Interestingly, the great majority of the responses to l-AP4 (95.4%) were observed in nerve terminals containing N-type channels. This specific co-localization of mGluR7 and N-type Ca2+-channels could explain the failure of the receptor to inhibit the P/Q channel-associated release component and also reveal the existence of specific targeting mechanisms to localize the two proteins in the same nerve terminal subset.     

The inhibition of glutamate release by metabotropic glutamate receptor 7 affects both [Ca2+]c and cAMP: evidence for a strong reduction of Ca2+ entry in single nerve terminals

Metabotropic glutamate receptors (mGluRs) from group III reduce glutamate release. Because these receptors reduce cAMP levels, we explored whether this signaling pathway contributes to release inhibition caused by mGluRs with low affinity for L-2-amino-4-phosphonobutyrate (L-AP4). In biochemical experiments with the population of cerebrocortical nerve terminals we find that L-AP4 (1 mm) inhibited the Ca(2+)-dependent-evoked release of glutamate by 25%. This inhibitory effect was largely prevented by the pertussis toxin but was insensitive to inhibitors of protein kinase C bisindolylmaleimide and protein kinase A H-89. Furthermore, this inhibition was associated with reduction in N-type Ca(2+) channel activity in the absence of any detectable change in cAMP levels. In the presence of forskolin, however, L-AP4 decreased the levels of cAMP. The activation of this additional signaling pathway was very efficient in counteracting the facilitation of glutamate release induced either by forskolin or the beta-adrenergic receptor agonist isoproterenol. Imaging experiments to measure Ca(2+) dynamics in single nerve terminals showed that L-AP4 strongly reduced the Ca(2+) response in 28% of the nerve terminals. Moreover, immunochemical experiments showed that 25-35% of the nerve terminals that were immunopositive to synaptophysin were also immunoreactive to the low affinity L-AP4-sensitive mGluR7. Then, mGluR7 mediates the inhibition of glutamate release caused by 1 mm L-AP4, primarily by a strong inhibition of Ca(2+) channels, although high cAMP uncovers the receptor ability to decrease cAMP.     

Aromatic trap analysis of free radicals production in experimental collagen-induced arthritis in the rat: protective effect of glycosaminoglycans treatment

Many findings demonstrated that Glycosaminoglycans (GAGs) and Proteoglycans (PGs) possess antioxidant activity. Collagen-induced arthritis (CIA) is an experimental animal model similar to human rheumatoid arthritis (RA) in which free radicals are involved. Sodium salicylate can be used as a chemical trap for hydroxyl radicals (OH •), the most damaging reactive oxygen species (ROS), yielding 2,5-dihydroxybenzoic acid), (2,5-DHBA) and 2,3-dihydroxybenzoic acid (2,3-DHBA). The measurement of these two acids in the plasma allows to indirectly assess the production of OH • radicals. The aim of the study was to investigate the effect of hyaluronic acid (HYA) (30 mg/kg i.p.) or chondroitin-4-sulphate (C4S) (30 mg/kg i.p.), on free radical production in Lewis rats subjected to CIA. After the immunization with bovine collagen type II in complete Freund's adjuvant, rats developed an erosive hind paw arthritis, that produced high plasma OH • levels assayed as 2,3-DHBA and 2,5-DHBA, primed lipid peroxidation, evaluated by analyzing conjugated dienes (CD) in the articular cartilage; decreased the concentration of endogenous vitamin E (VE) and catalase (CA) in the joint cartilage; enhanced macrophage inflammatory protein-2 (MIP-2) serum levels and increased elastase (ELA) evaluated as an index of activated leukocyte polymophonuclear (PMNs) accumulation in the articular joints. The administration of HYA and C4S starting at the onset of arthritis (day 11) for 20 days, limited inflammation and the clinical signs in the knee and paw, reduced OH • production, decreased CD levels, partially restored the endogenous antioxidants VE and CA, reduced MIP-2 serum levels and limited PMNs infiltration. The results indicate that the GAGs HYA and C4S significantly reduce free radical production in CIA and could be used as a tool to investigate the role of antioxidants in RA.