Jean-Pierre Ballini

An Ultraviolet Micro-Raman Spectrometer: Resonance Raman Spectroscopy within Single Living Cells

Ultraviolet-excited resonance Raman spectra have been obtained for the first time for microsamples of dAMP deoxymononucleotide and DNA and for the nuclear DNA of a single living T47D cultured cell. The spectral variations observed in changing the excitation wavelength reflect the dramatic variations of the Raman cross sections of the various DNA compounds in the ultraviolet region. Many of the spectral features of the single-cell nuclear DNA excited by 257-nm radiation essentially arise from cellular purines, although a contribution from the protein amide I band and from the tryptophan residues is probably observed in the 1600–1700 cm−1 spectral region.

Microspectrofluorimetric study of the kinetics of cellular uptake and metabolization of benzo(a)pyrene in human T 47 D mammary tumor cells: evidence for cytochrome P1450 induction

The kinetics of penetration, activation and detoxification of benzo(a)pyrene were determined by near U.V. microspectrofluorimetric measurements on single living cells. This technique allows one to monitor the different intracellular fluorescent species present in a subcellular microvolume by using spectral decomposition of the fluorescence data. The T47-D cell line was chosen for its high capability of metabolization. The penetration involves a simple diffusion transfer through the cytoplasmic membrane of the cell, with a half-time of ∼ 2 min. The metabolization process gives rise, with more than a one hour delay after intracellular incorporation of the hydrocarbon, to a rapid conversion of B(a)P into unconjugated metabolites, leading to a transient accumulation of the 3OH-B(a)P metabolite in the cell. This feature may be related to the enhancement of cytochrome P1450 activity, induced by the B(a)P itself. The ability of the cell to increase its Cyt-P1450 level, after exposure to B(a)P, gives indirect evidence for the presence of the Ah gene complex in the T47-D cell line.

Wavelength-resolved lifetime measurements of emissions from DNA components and poly rA at room temperature excited with synchrotron radiation

Abstract The wavelength-resolved luminescence decays of the very weak emission from DNA and of some components at room temperature have been investigated by using the luminescence equipment recently built at Orsay. It combines the LURE synchrotron source with a spectrophotofluorometer using a fast single photon counting detection operating in the nanosecond range. The high intensity of the synchrotron source together with the high pulse frequency have allowed for the first time the luminescence decays of such weak emitters (φf ⩽10-4) to be undertaken. It is shown that the room temperature fluorescence of the monomers (nucleosides) is indeed very short, τ

FORMATION OF CYCLOBUTANE THYMINE DIMERS PHOTOSENSITIZED BY PYRIDOPSORALENS: A TRIPLET-TRIPLET ENERGY TRANSFER MECHANISM

Abstract— The 365 nm irradiation of thymine thin films in the presence of pyridopsoralens is shown to induce the formation of cyclobutane thymine dimers, in contrast to other compounds such as 8‐and 5‐methoxypsoralen. In order to elucidate the mechanism of such a photosensitized reaction, we have determined the energy of the lowest triplet state (T,) of these compounds, using phosphorescence spectroscopy and CNDO/S quantum chemistry calculations. The T, energy values were found to be significantly higher for pyridopsoralens–up to 0.3 eV–than for 8‐ and 5‐methoxypsoralen (approximately 2.8 eV), which are not able to photoinduce cyclobutane thymine dimers. The determination of the relative efficiency of cyclobutane thymine dimer formation was performed using chromatographic analysis. A good correlation was found between the energy of the T, state of the psoralen derivatives and the related cyclobutane thymine dimer formation. Moreover, the photosensitized cyclobutane thymine dimer formation appeared to be temperature‐dependent. Our results are consistent with a mechanism involving a triplet energy transfer from the pyridopsoralen to thymine.

GEOMETRY OF INTERCALATION OF PSORALENS IN DNA APPROACHED BY MOLECULAR MECHANICS

The results of molecular mechanical calculations on intercalation complexes of 3‐car‐bethoxypsoralen, 5‐methoxypsoralen, 8‐methoxypsoralen, 7‐methylpyrido[3,4‐c]psoralen (MepyPs) and 7‐methylpyrido[4,3‐c]psoralen (2N‐MePyPs) with the double stranded duodecanucleotide d(CGCGATATCGCG)2 are presented. In the energy‐minimized structures, the psoralens are intercalated with their plane orthogonal to the helix axis. Stacking interactions between the furan ring of the psoralen and the adjacent bases are maximized in most derivatives studied, whereas the effect of the various substituents of the psoralen ring is to specifically push part of the molecule towards either the minor or the major groove, preventing a symmetrical intercalation (with respect to the two strands of the DNA). The relative position of the psoralen ring and of the adjacent thymine foreshadows the formation of furan‐side monoadducts in 3‐CPs, MePyPs and 2N‐MePyPs, whereas the formation of a pyrone‐side monoadduct appears as geometrically more favourable in 5‐MOP and both furan‐ and pyrone‐side monoadducts can be geometrically envisaged in 8‐MOP. A good correlation therefore exists between the more or less favourable equilibrium geometries and the experimentally observed photoreactions. The present study is the first attempt to characterize the geometrical parameters as part of a complex set of geometrical, dynamical and excited state parameters governing the overall DNA‐psoralen photoreaction.

Time-resolved fluorescence emission and excitation spectroscopy of d(TA) and d(AT) using synchrotron radiation

The photophysics of the sequence isomers d(TA) and d(AT) has been investigated at room temperature in 5 x 10(-5) M neutral aqueous solution using pulsed ultraviolet excitation from the ACO synchrotron and detection by time correlation or gated single-photon counting. Decay profiles of the emissions at 350, 400 and 460 have been analyzed both independently and globally by reiterative non-linear least-squares fitting to models of two and three independently emitting species. No evidence has been observed for excited-state reaction. Time-windowed spectra, both emission and excitation, have been collected for three time windows and have been deconvoluted to give time-resolved spectra using the lifetimes resulting from the decay analyses. Spectra are separated into two classes, with picosecond and nanosecond lifetimes, respectively. The picosecond spectra have the emission and excitation spectral characteristics of mixed monomer (A and T) fluorescences and are assigned as originating from the unstacked fractions of d(TA) and d(AT). The nanosecond emission spectra from d(TA) and d(AT) are both two-component, with lambda max approximately 350 and approximately 425 nm and lifetimes of 2.3 and 6.1 ns, respectively. The time-resolved excitation spectra for the nanosecond emissions are quite different from the isotropic absorption spectra of d(TA) and d(AT) but correlate with the anisotropic absorption for out-of-plane transitions between stacked bases of co-crystals of 9-methyladenine and 1-methylthymine reported by Stewart and Davidson. The nanosecond spectra thus represent the direct excitation and emission of stacked pairs of bases. These results provide no evidence for energy transfer and are probably related to sequence-specific photo-adduct formation.

Spectres d'impulsions à ⪡un photoélectron⪢ des photomultiplicateurs

Abstract Analysing the histogram of anode pulse amplitudes allows a discussion of the hypotheses that have been proposed to account for the statistical processes of secondary multiplication in a photomultiplier (PM 56 DVP): a Poisson process, as was suggested by Lombard and Martin (1961), or a Polya process, as suggested by Prescott (1966). Comparison of experimental single-electron pulse spectra with calculated theoretical spectra is first performed by means of moment analysis, from which fractional variance, skewness (a measure of the asymmetry of the distribution), and excess of kurtosis (a measure of the sharpness of the peak and the extent of the tails, as compared with those of a Gaussian distribution), are deduced; then, by directly comparing the shapes of the spectra and minimizing the area between them. On the whole, this study on a focussed dynodes PM makes it clear that though a Polya process is a better approach to reality than a Poisson process, the gain distribution which it leads to does not correspond to the experimental spectrum. A better accord is obtained on assuming a dynode distribution including, in a first approximation, two Poisson distributions of distinct mean value and expressing an inhomogeneity of the secondary multiplication gain value different from that imagined by Prescott.

Molecular mechanics and dynamics of DNA—furocoumarin complexes: Effect of the aromatization of the pyrone ring on the intercalation geometry☆

Results of molecular mechanics and dynamics calculations on intercalation complexes of DNA with various furocoumarins (psoralen, angelicin, 7-methylpyrido[3,4-c]psoralen and 7-methylpyrido[4,3-c]psoralen) and their corresponding aromatized derivatives are presented. These calculations were undertaken with the aim to elucidate the roles of the pyrone and pyridine moieties in the interactions which tend to orient the furocoumarins and pyridopsoralens between DNA base pairs. It appears that the intercalation geometries are very similar for the furocoumarins and related aromatized compounds. Therefore the oxygen and nitrogen atoms of the pyrone and pyridine moieties are not important in the orientation of the drug within the oligonucleotide.

Molecular mechanics and dynamics study of DNA-furocoumarins complexes: Effect of methylation of the angular derivatives on the intercalation geometry

SummaryResults of molecular mechanics calculations on intercalation complexes between DNA and angelicin derivatives: angelicin, 4′-methylangelicin, 5′-methylangllicin, 4,4′-dimethylangelicin, 4,5′-dimethylangelicin, 4,6,4′-trimethylangelicin and 4,6,5′-trimethylangelicin, are presented. The correlation between the presence of methyl groups and an increase in DNA photobinding affinity is discussed on the basis of the molecular structures. The influence of the orientation of the angelicins within the intercalation cavity is also discussed. Finally, the consequences of the dynamical behaviour of angelicin in the intercalation cite are studied.

A Conformational Study of Lys-Arg-Asp-Ser and Analogs, a Series of Potent Antithrombotic Peptides. An Approach Based on Simulated Annealing and 1H NMR

Simulated annealing techniques were used to explore the conformational space of the potent antithrombotic peptide L.Lys-L.Arg-L.Asp-L.Ser (KRDS) and of two analogs: D.Lys-L.Arg-L.Asp-L.Ser (KDRDS), which is inactive, and L.Lys-L.Arg-L.Glu-L.Glu (KREE), which exhibits a strong biological activity. For each peptide, a set of initial conformations was generated and submitted to simulated annealing, including a heating to 1000 K followed by a cooling to 300 K. 200 resulting conformations of each compound were analyzed and classified according to the network of electrostatic interactions involving charged side chains and charged C- and N-terminal groups. A reduced number of conformational classes was obtained and conformations corresponding to predominant classes were found to be in qualitative agreement with structural parameters deduced from 1H NMR spectra. A comparison between the classes of the active and non active peptide was achieved. Some conformations were found to be specific of active peptides.