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101.
Diana Over Gildas Bertho Miguel-Angel Elizondo-Riojas Jiří Kozelka 《Journal of biological inorganic chemistry》2006,11(2):139-152
The bulky, asymmetric analog of the antitumor drug cisplatin, [PtCl(2)(tmen)] (tmen = N,N,N'-trimethylethylenediamine), was used to produce crosslinks with the dinucleotide d(GpG), modeling the most frequent lesions that cisplatin and its analogs cause to DNA. The ligand tmen was chosen because it is expected to constrain the guanine cis to the NMe(2) group in the adduct [Pt(tmen){d(GpG)}](+) to an orientation perpendicular to the coordination plane and to stabilize the other guanine in an oblique orientation, thus maintaining a head-to-head geometry typical of cisplatin-d(GpG) crosslinks within single- and double-stranded DNA. Of the four possible combinations of tmen chirality (R or S symmetry of the coordinated NHMe group) and crosslink direction (5'-G bound cis to the secondary or the tertiary amino group of tmen), two isomers were preponderantly formed, [Pt(R-tmen){d(GpG)}](+) with 5'-G bound cis to NMe(2) and [Pt(S-tmen){d(GpG)}](+) with 5'-G bound cis to NHMe. The former was shown to have a right-handed R2 orientation of guanines similar to that found in duplex DNA, whereas the latter had a left-handed L1 orientation that modeled cisplatin-d(GpG) adducts within single-stranded DNA. The R2 rotamer was found to be in an equilibrium (as observed using EXSY spectroscopy) with a minor fraction (< or =4%) of a Delta-HT rotamer related to R2 by rotation of the 3'-G about the Pt-N7 bond. The major rotamers R2 and L1 were isolated using reverse-phase HPLC, and their NMR and CD signatures were compared to those of the corresponding rotamers of the less hindered adduct [Pt(dmen)(GpG)](+) (dmen = N,N-dimethylethylenediamine). From this and other comparisons with previously reported platinum dinucleotide complexes, and from molecular modeling, it could be concluded that both steric repulsion between guanine and substituents of the cis amino group and N-H...O6 hydrogen bonding are significant effects favoring the oblique orientation of one guanine base typical of the HH rotamers of [Pt(diamine){d(GpG)}](+) and [Pt(diamine)(GpG)](+) complexes. 相似文献
102.
Frank S. Choveau Aziza El Harchi Nicolas Rodriguez Bénédicte Louérat-Oriou Isabelle Baró Sophie Demolombe Flavien Charpentier Gildas Loussouarn 《Biophysical journal》2009,97(5):1323-1334
Studies in Shaker, a voltage-dependent potassium channel, suggest a coupling between activation and inactivation. This coupling is controversial in hERG, a fast-inactivating voltage-dependent potassium channel. To address this question, we transferred to hERG the S3-S4 linker of the voltage-independent channel, rolf, to selectively disrupt the activation process. This chimera shows an intact voltage-dependent inactivation process consistent with a weak coupling, if any, between both processes. Kinetic models suggest that the chimera presents only an open and an inactivated states, with identical transition rates as in hERG. The lower sensitivity of the chimera to BeKm-1, a hERG preferential closed-state inhibitor, also suggests that the chimera presents mainly open and inactivated conformations. This chimera allows determining the mechanism of action of hERG blockers, as exemplified by the test on ketoconazole. 相似文献
103.
Vincent Gueret Juan A. Negrete-Virgen Andrew Lyddiatt Mohamed Al-Rubeai 《Cytotechnology》2002,38(1-3):87-97
There is a constant and growing interest in exploitingadenoviruses as vectors for gene therapy when transientexpression of a therapeutic protein is necessary. Therequirement for an increased viral titre has prompted asearch for techniques by which this virus may be assayedwith greater speed and simplicity. Conventional plaqueassay for quantification of adenoviral vectors titre incurrent use is laborious and time-consuming (up to 14days). We report herein a method for the monitoring ofadenovirus expressing green fluorescent protein thatincorporates rapid and easy sample handling by means offlow cytometric analysis. Cells (HEK293) were infectedwith adenovirus at various multiplicity of infection(MOI), harvested 17 to 20 h post infection and analysedby flow cytometry. Assumptions were made that onefluorescent cell was infected by a single infectiousparticle at a relatively low MOI. The adenoviral titrewas subsequently estimated from cell analysis in arelatively short time. The results obtained with an E1-complementing cell line (HEK293) were compared with thatobtained using a non-complementing cell line (A549). APoisson distribution successfully modelled the profile ofinfection as a function of MOI. This provided a betterunderstanding of adenoviral infection at the earlieststage possible. Monitoring of GFP fluorescence and viruspropagation in a batch culture of infected cells wassubsequently used as a practical application of thevalidated method. 相似文献