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

2000

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Domain motions in proteins

New numerical techniques permit the identification and characterization of dynamical domains in proteins with almost negligible computational effort. The validity of the approximations on which these techniques are based provides valuable insight into the nature of the energy landscape of proteins. An easy-to-use interactive analysis program based on the new methods is available. Extensions to higher frequency ranges are discussed briefly. (C) 2000 Elsevier Science B.V. All rights reserved.

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Dynamics of a double stranded DNA oligomer : Mode-coupling diffusion approach and reduced rigid fragment models

The local dynamics of a double stranded DNA fragment [d(CpGpCpApApApTpTpTpGpCpG)](2) of twelve base pairs is ;obtained to second order in the mode-coupling expansion of the Smoluchowski diffusion theory. The DNA is considered a fluctuating three-dimensional (3D) structure undergoing rotational diffusion. The starting structure for the calculations is the B canonical structure of the fragment, while the fluctuations are evaluated using molecular dynamics simulations, with the ensemble averages approximated by time averages along a trajectory of length 1.5 ns.

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Dynamics of HIV-specific CD8(+) T lymphocytes with changes in viral load

The influence of HIV burden variations on the frequencies of Ag-specific CD8(+) T cell responses was evaluated before and during highly active antiretroviral therapy by analyzing the number, diversity, and function of these cells. The frequencies of HLA-A2-restricted CD8+ PBL binding HLA-A2/HIV-epitope tetramers or producing IFN-gamma were below 1%, A panel of 16 CTL epitopes covering 15 HLA class I molecules in 14 patients allowed us to test 3.8 epitopes/patient and to detect 2.2 +/- 1.8 HIV epitope-specific CD8(+) subsets per patient with a median frequency of 0.24% (0.11-4.79%).

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Effect of ethidium bromide intercalation on DNA radiosensitivity

Purpose : To assess the influence of the intercalating drug ethidium bromide (EtBr) on the yields of single strand breaks (ssb) induced by fast neutrons in supercoiled pBR322 plasmid and in a linear DNA restriction fragment. Materials and methods : The yield of ssb in the plasmid was measured by agarose gel electrophoresis. The proportion of fragments bearing one ssb and the probability of breakage at each nucleotide site was determined using sequencing gel electrophoresis. The volume variations due to the intercalation of EtBr were calculated. The expected radio-modifying effect at each nucleotide site of the linear fragment was evaluated using a reported simulation procedure.

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Efficient gene transfer into human normal and cystic fibrosis tracheal gland serous cells with synthetic vectors

Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of re porter gene transfer into immortalized normal (MM-39) and CF (CF-KM I) human airway epithelial gland serous cells using various synthetic vectors : glycosylated polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using a-glycosylated polylysine, 20% of the cells were transfected. At 24 h after CF trans membrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using a-glycosylated poly lysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane.

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EPR on aqueous Gd3+ complexes and a new analysis method considering both line widths and shifts

We performed variable temperature (0-100 degrees C), concentration and frequency (9.425, 75, 150 and 225 GHz) continuous wave electron paramagnetic resonance (EPR) measurements on three different Gd(III) compounds : [Gd(H2O)(8)](3+), [Gd(DOTA)(H2O)](-) (DOTA : 1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane) and [Gd(DTPA-BMA)(H2O)] (DTPA-BMA : 1,5-[bis(N-methylcarbamoyl)methyl]-1,3,5-tris(carboxymethyl)-1,5-diamino-3-azapentane) in aqueous solution. A simultaneous analysis of peak-to-peak widths and dynamic frequency shifts provides access to the transverse electronic relaxation, which is described using a transient zero field splitting (ZFS) mechanism with a spin rotation contribution. Our simultaneous analysis procedure involves numerical calculations using the full relaxation matrix and yields results in acceptable agreement with experimental data for reasonable values of the ZFS parameters (trace of the square of the ZFS Hamiltonian Delta(2)=10(19)-10(20) s(-2) depending on the complex, correlation time of the fluctuations tau(v)(298)=10(-11)-10(-10) s). We also discuss the relationship between our approach and recent developments found in the literature.

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Equilibria and formation kinetics of some cyclen derivative complexes of lanthanides

The kinetics of the formation reactions between the lanthanide(III) ions Ce3+, Eu3+ and Yb3+ and four cyclen derivative ligands, DO3A-B, DO3A-ME, DO2A and DO2A-2B, were studied by spectrophotometry and a stopped-flow method at 25 degrees C in 1.0 M KCl solutions. The reactions were found to be of first order, which was interpreted in terms of the formation of a diprotonated intermediate, Ln(H2L)(+). The formation of products occurs via deprotonation and rearrangement of the intermediate, characterised by the rate constant, ii,. The rate law obtained, k(r) = k(OH)[OH ], is similar to those obtained for the formation reactions of DOTA and DOTA derivative complexes. The rate constants, k(OH), decrease with decrease in the number of charged carboxylate functional groups in the ligandsl the lowest rates were found for the formation of the DO2A complexes.

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Evidence for myocardial ATP compartmentation from NMR inversion transfer analysis of creatine kinase fluxes

The interpretation of creatine kinase (CK) flux measured by P-31 NMR magnetization transfer in vivo is complex because of the presence of competing reactions, metabolite compartmentation, and CK isozyme localization. In the isovolumic perfused rat heart, we considered the influence of both ATP compartmentation and ATP-P-i exchange on the forward (F-f : PCr —> ATP) and reverse (F-r) CK fluxes derived from complete analysis of inversion transfer. Although F-f should equal F-r because of the steady state, in both protocols when PCr (inv-PCr) or ATP (inv-ATP) was inverted and the contribution of ATP-P-i was masked by saturation of P-i (sat-P-i), F-f/F-r significantly differed from 1 (0.80 +/- 0.06 or 1.32 +/- 0.06, respectively, n = 5). These discrepancies could be explained by a compartment of ATP (f(ATP)) not involved in CK. Consistently, neglecting ATP compartmentation in the analysis of CK in vitro results in an underestimation of F-f/F-r for inv-PCr and its overestimation for inv-ATP. Both protocols gave access to f(ATP) if the system was adequately analyzed. The fraction of ATP not involved in CK reaction in a heart performing medium work amounts to 20-33% of cellular ATP. Finally, the data suggest that the effect of sat-P-i might not result only from the masking of ATP-P-i exchange.

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