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Accueil > Publications > Recherche par années > Années 2000 > 2000

2000

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Hybrid and complex glycans are linked to the conserved N-glycosylation site of the third eight-cysteine domain of LTBP-1 in insect cells

Covalent association of LTBP-1 (latent TGF-beta binding protein-1) to latent TGF-beta is mediated by the third eight-cysteine (also referred to as TB) module of LTBP-1, a domain designated as CR3. Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3, and we show here that another lepidopteran cell line, Trichoplusia ni TN-SB 1-4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3. CR3 contains an N-glycosylation site, which is conserved in all forms of LTBP known to date. When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells.

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Identification of by-products from an orthogonal peptide ligation by oxime bonds using mass spectrometry and tandem mass spectrometry

Synthetic proteins with unusual architecture are obtained through chemoselective ligation, a method based on the condensation of unprotected peptides under mild aqueous conditions,. The last step of a new procedure leading to a tri-branched conjugate consists of the chemoselective ligation reaction between an (amioooxy)acetyl peptide and a peptide aldehyde resulting from a first ligation via an oxime bond. In order to optimize the reaction conditions, electrospray ionization mass spectrometry combined with Liquid chromatography and tandem mass spectrometry has been used.

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Identification, purification and partial characterisation of an oligonucleotide receptor in membranes of HepG2 cells

The low and unpredictable uptake and cytosolic transfer of oligonucleotides (ODN) is a major reason for their limited benefit. Improving the ODN potential for therapy and research requires a better understanding of their receptor-mediated endocytosis. We have undertaken to identify a membrane ODN receptor on HepG2 cells by ligand blotting of cell extracts with [I-125]ODN and by photolabelling of living cells with a [I-125]ODN-benzophenone conjugate. A major band at 66 kDa was identified by the two methods.

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In vitro evidence for the interaction of tRNA(3)(Lys) With U3 during the first strand transfer of HIV-1 reverse transcription

Over the course of its evolution, HIV-1 has taken maximum advantage of its tRNA(3)(Lys) primer by utilizing it in several steps of reverse transcription. Here, we have identified a conserved nonanucleotide sequence in the U3 region of HIV-1 RNA that is complementary to the anticodon stem of tRNA(3)(Lys). In order to test its possible role in the first strand transfer reaction, we applied an assay using a donor RNA corresponding to the 5’-part and an acceptor RNA spanning the 3’-part of HIV-1 RNA. In addition, we constructed two acceptor RNAs in which the nonanucleotide sequence complementary to tRNA(3)(Lys) was either substituted (S) or deleted (Delta).

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Lanthanide podates with programmed intermolecular interactions : Luminescence enhancement through association with cyclodextrins and unusually large relaxivity of the gadolinium self-aggregates

The synthesis of the phenyl anchored podand H4L1 fitted with four 3-carboxylate pyrazole arms and programmed for intermolecular interactions is reported, and its protonation constants are determined. interaction with Ln(3+) ions (Ln = La, Eu, Lu) in dilute aqueous solutions leads to complexes with 1:I and 1:2 metal-ligand stoichiometry. The stability constants are in the range log beta (110) = 12.7-13.5 and log beta (120) = 22.5-23.8 (pLn values in the range 9-10). The podates display a fair sensitization of the metal-centered luminescence with an absolute quantum yield of 5% in case of Tb-III. The average numbers of water molecules coordinated to the Ln(III) ion amount to 3.8 and 4.9 for the 1:1 Eu and Tb podates, respectively, as determined by lifetime measurements.

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