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Accueil > Publications > Recherche par années > Années 1990 > 1999

1999

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Intracellular localization of oligonucleotides : Influence of fixative protocols

In many studies reporting the use of antisense oligonucleotides (ODN), the intracellular localization was investigated by using fluorescent-labeled oligonucleotides (F-ODN), More often, cells were fixed on uptake of F-ODN before microscopic analysis. We report here the influeuce of various methods of cell fixation on the intracellular localization of ODN, By confocal microscopy, we show that with unfixed cells, endocytosed peptides, oligonucleotides (M-r around 10,000), and endocytosed proteins were mainly localized in vesicular compartments. On mild fixation with paraformaldehyde, an identical intracellular localization was observed repeatedly after fixation, from immediately up to several days, In contrast, with methods based on the use of strong fixatives, such as methanol or acetone, the small molecules diffuse into the cytosol and in the case of oligonucleotides into the nucleus. These results point out the importance of the fixation protocol in the study of intracellular localization of ODN and their derivatives.

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Intradermal DNA immunization : Antisera specific for the membrane lectin MR60/ERGIC-53

The trafficking of intracellular membrane proteins in Golgi apparatus, endoplasmic reticulum or intermediate compartment has not yet been fully elucidated. The human MR60/ ERGIC-53 and the rat p58 proteins are one such protein ; and to study them in cell-free and in situ systems, high quality monospecific antisera are required. Highly specific antisera have been obtained after immunization of mice with plasmids containing a gene encoding either the full length or a truncated protein. The best results were obtained after intradermal injections of a plasmid encoding a truncated protein comprising both the luminal carbohydrate recognition domain and the stem down to a cysteine residue close to the C-terminal end, but neither the transmembrane nor the cytosolic domains. Such antisera have a very high titer and are very efficient tools to visualize the MR60 protein in situ or to selectively precipitate the MR60 proteins from a whole cell lysate.

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Investigation of subsite preferences in aminopeptidase A (EC 3.4.11.7) led to the design of the first highly potent and selective inhibitors of this enzyme

The study of the physiological roles of the membrane-bound zinc-aminopeptidase A (glutamyl aminopeptidase, EC 3.4.11.7) needs the design of efficient and selective inhibitors of this enzyme. An acute exploration of aminopeptidase A active site was performed by a combinatorial approach using (3-amino-2-mercapto-acyl)dipeptides able to sc its S-1, S-1’, and S-2’ subsites. This analysis confirmed that the S-1 subsite is optimally blocked by a glutamate or isosteric residues and demonstrated that the S-1’ subsite is hydrophobic whereas the S-2’ subsite recognizes preferentially negatively charged residues derived from aspartic acid. The optimization of these structural parameters led to the synthesis of nanomolar and subnanomolar inhibitors of aminopeptidase A such as H3N+CH(CH2CH2SO3-)CH(SH)CO-Ile-(3-COOH)Pro that exhibits a K-i of 0.87 nM. The best compounds were synthesized by a stereochemically controlled route. These first described highly potent inhibitors could allow studies about the role of physiological substrates of APA such as angiotensin II- and cholecystokinin CCK8 in the central nervous system.

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Lanthanide(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complexes in acidic medium : significant decrease in water exchange rate

UV-Vis and lanthanide-induced O-17 shift measurements on the complex [Eu(DOTA)](-) (H(4)DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) have shown that the inner co-ordination sphere of the Ln(3+) ion is not affected on protonation which suggests that the proton is attached to a non-co-ordinated oxygen atom of a carboxylate group. Proton NMR measurements performed as a function of the H+ concentration revealed that the protonation slightly accelerates the intramolecular dynamic processes : the enantiomerization for [La(DOTA)](-) and the enantiomerization and interconversion between the major and minor isomer for [Eu(DOTA)](-). Contrary to first glance expectations, the water exchange rate on [Gd(DOTA)(H2O)](-) decreases significantly with increasing extent of protonation, and at 1.0 M H+ concentration is about ten times lower than in neutral media. In 1.0 M acidic solution the proton relaxivities were found to be higher than expected solely on the basis of the water exchange rates. This finding is interpreted with a faster proton exchange in acidic solutions which is the consequence of the catalytic effect of H+ ions.

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Leukemic CD3(+) LGL share functional properties with their CD8(+)CD57(+) cell counterpart expanded after BMT

Leukemic T-LGL (large granular lymphocyte) composed of clonal CD3(+)TCR alpha beta(+)CD8(+)CD57(+) cells were compared with oligoclonally CD3(+)CD8(+)CD57(-) lymphocytes expanded after BMT. Leukemic CD3(+)CD8(hl+)CD57(+) LGL showed several phenotypic differences such as an upregulation of CD16 and adhesion molecules (mainly CD11c, CD58 and CD54), activation markers and an exclusive CD45RA isoform expression. Unstimulated CD3(+)CD8(+)CD57(+) LGL from both leukemic and BMT donors spontaneously developed an ex vivo CTL-like CD3-redirected cytotoxicity but no NK cell activity. Different stimuli (PHA, PMA or rhlL-2) induced similar cytotoxic profiles after a 6-day culture involving a CD3-redirected lysis predominating over a low NK cell activity.

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Lichenysins G, a novel family of lipopeptide biosurfactants from Bacillus licheniformis IM 1307 : Production, isolation and structural evaluation by NMR and mass spectrometry

A series of 9 lactonic lipopeptide biosurfactants was isolated from Bacillus licheniformis IM 1307 as representatives of the lichenysin group and we propose to name them lichenysins G. They were recovered from the culture medium as complex mixtures of molecules having different peptide sequences and different structures of P-hydroxy fatty acids. Their separation was achieved by a reversed-phase HPLC method leading to eight well-separated compounds. The complete structure of individual isoforms was proposed following the results of amino acid and fatty acid analysis, LSI-MS and 2D NMR spectroscopies. Compared to surfactin, lichenysins G are at least 10 fold more efficient biosurfactants.

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Life in the solar system

Life, defined as a chemical system capable of transferring its molecular information via self-replication and also capable of evolving, must develop within a liquid to take advantage of the diffusion of complex molecules. On Earth. life probably originated from the evolution of reduced organic molecules in liquid water. Organic matter might have been formed in the primitive Earth’s atmosphere or near hydrothermal vents. A large fraction of prebiotic organic molecules might have been brought by extraterrestrial-meteoritic and cometary dust grains decelerated by the atmosphere. Any celestial body harboring permanent liquid water may therefore accumulate the ingredients that generated life on the primitive Earth.

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Metallopeptidase inhibitors of tetanus toxin : A combinatorial approach

The bacterial protein tetanus toxin (TeNt), which belongs to the family of zinc endopeptidases, cleaves synaptobrevin, an essential synaptic protein component of the neurotransmitter exocytosis apparatus, at a single peptide bond (Gln(76)-Phe(77)). This protease activity is a particularly attractive target for designing potent and selective synthetic inhibitors as a possible drug therapy for tetanus. ß-Aminothiols mimicking Gln(76) of synaptobrevin have been previously shown to inhibit the tetanus neurotoxin enzymatic activity in the 35-250 mu M range. These compounds have now been modified to interact with S’ subsites of the TeNt active site, with the aim of increasing their inhibitory potencies. Combinatorial libraries of pseudotripeptides, containing an ethylene sulfonamide or an m-sulfonamidophenyl moiety as the P-1 side chain and natural amino acids in P-1’ and P-2’ positions, were synthesized. The best inhibitory activity was observed with Tyr and His as P-1’ and P-2’ components, respectively. This led to new inhibitors of TeNt with K-i values in the 3-4 mu M range. These molecules are the most potent inhibitors of TeNt described so far.

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