Influence of oxygen on the radiochemical inactivation of the microsomal monooxygenase complex.
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Animals↗
Biomedical subjects
V Favaudon
Publications and source records attributed to V Favaudon.
Kinetics of reduction of the cytochrome C3 from Desulvovibrio vulgaris.
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Cytochrome c Group↗
Structure of the oxygen adduct intermediate in the bacterial luciferase reaction: C nuclear magnetic resonance determination.
By using FMN enriched in (13)C (90%) at position C-4a, we have conclusively shown that the reaction of molecular oxygen with bacterial luciferase-bound FMNH(2) forms an adduct at the 4a position. Consistent with this are (13)C NMR studies of FMN and other flavin compounds which show that this carbon should be unusually reactive in the reduced 1,5-dihydroflavins with respect to electrophilic attacks.
Journal Article↗
Oxidation kinetics of 1,5-dihydroflavin by oxygen in non-aqueous solvent.
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Dimethylformamide↗
Magnesium dependence of the association kinetics of Escherichia coli ribosomal subunits.
The magnesium dependence of the Escherichia coli ribosomal subunits association has been investigated by the stopped-flow technique using isolated 30S and 50S particles depleted of polyamines and any initiation factor. Binding of the fluorescent probe bis(8-anilino-1-naphthalenesulfonate) to the ribosomal proteins occurs through biphasic kinetics. A dark reaction corresponding to a very rapid, reversible complexation of the dye molecule is followed by a slow photochemical reaction that gives rise to irreversible addition of the probe. Only the 30S subparticle exhibits a magnesium-dependent conformational change from the kinetic analysis of the dark reaction. The 70S formation kinetics are limited by a conformational change of the 30S subunit if this particle is depleted of Mg2+ (1 mM Mg2+/50 mM K+), while its activated structure is restored by incubation with 8 mM Mg2+/50 mM K+. No rate-limiting conformation rearrangement of the 50S subunit could ever be evidenced. The Mg2+ dependence of the association kinetics of preactivated ribosomal particles is satisfactorily explained by electrostatic effects and/or formation of salt bridges, in agreement with the results of Wishnia and co-workers (Wishnia, A. Boussert, A., Graffe, M., Dessen, P., and Grunberg-Manago, M. (1975), J. Mol. Biol. 93, 499). Equilibrium studies indicate that the ribosomal preparations we used are of B type, according to Debey et al. (Debey, P., Hui Bon Hoa, G., Douzou, P., Godefroy-Colburn, T., Graffe, M., AND Grunberg-Manago, M. (1975) Biochemistry 14, 1553). The addition of spermidine results in a drastic fall of the need of Mg2+ for association, but it does not allow conversion of B-type particles into A-type ones at 25 degrees C. In addition to that, some 30S-bound spermidine appears to be involved directly in the coupling reaction.
Anilino Naphthalenesulfonates↗
The kinetics of flavine oxidation--reduction. I. Dismutation in nonaqueous solvent.
The dismutation reactions of flavines in dimethylformanide have been investigated using the stopped-flow technique under anaerobic conditions. The ionization constants of fully reduced and oxidized tetraacetylriboflavine were measured spectrophotometrically in buffered dimethylformanide. The dismutation equilibrium of the flavine as a function of pH in dimethylformanide was roughly comparable to that reported in water and allowed the estimation of the pKa value of the flavosemiquinone. The dismutation kinetics of tetraacetylriboflavine in unbuffered dimethylformanide were investigated using the fully oxidized and reduced flavines in their neutral form at constant produce of concentrations and varying the reduction degree. The kinetics at very low reduction ratios (less than3%) were triphasic. The kinetic analysis of the initial and simultaneous formation of the anionic and neutral radicals revealed a second-order reaction. The electron transfer between the oxidized and reduced flavines was not directly coupled with prton exchange. The multiphasic time course of the reaction proceeded primarily from differences in the intrinsic rates of the direct and mixed backward dismutation reactions of the two radical species, and finally from a change in the equilibrium conditions resulting from the accumulation of anionic flavohydroquinone. An acidic-basic negative catalytic effect from the neutral flavohydroquinone appeared progressively as the reduction degree was increased. It was complete at reduction ratios higher than 30%, i.e. under conditions where the radical anion could not be observed at any reaction time. Acids with a pKa value lower than the second one of the flavosemiquinone exhibited a similar catalytic effect. These acidic-basic catalytic effects are associated with changes in the ionic state of labile intermediate dimers formed in the forward as well as in the backward direactions of the dismutation reaction. Such a transient complex revealed by the kinetic analysis could be observed directly by absorption spectroscopy in alkaline-buffered dimethylformanide. Its spectral characteristics, as well as the kinetic effects induced by substitution of the benzenoid part of the flavine, can hardly be taken into account by a quinhydrone-like structure for the intermediate dimers at any pH value. The experimental results favored a more specific interaction, possibly of covalent character, involving the benzenoid part of the isoalloxazine ring.
Chemical Phenomena↗
The kinetics of flavine oxidation-reduction. II. Metal ion interactions.
The oxidation-reduction reactions of tetraacetylriboflavine in the presence of various metal ions in dimethylformamide have been investigated using the stopped-flow technique under anaerobic conditions. Dismutation kinetics in the presence of redox-inactive dissociated divalent metal ions such as Cd2+, Zn2+, and Fe2+ are typically triphasic. Metal ions act primarily upon an intermediate flavine dimer formed by fast association of flavoquinone and flavohydroquinone, resulting in a parallel formation and neutral and chelated radicals. A competition between metal ions and proton donors, e.g. the neutral flavohydroquinone (FredH3), is observed at the level of this intermediate complex. Small spectral changes occur secondarily as an ill-defined intermediate phase which could correspond to the reorganization of the solvation of radical chelate. The neutral radical is finally chelated at a much slower rate, the yield of total radical formation remaining almost unchanged during this kinetic phase. The oxidation of flavohydroquinone by ferric ions, either dissociated or strongly coordinated within a porphyrin, is complete and proceeds through biphasic kinetics. The first phase (Fred leads to F) is much faster than the second one (F leads to Fox). Dismutation resulting from the transient accumulation of neutral flavosemiquinone competes with the direct oxidation with ferric ions for the completion of the second oxidation step. The relative rate of dismutation is essentially limited by acidic-basic reactions in the absence of an excess of ferrous ion. The kinetic analysis of the direct oxidation reactions favors an outer-sphere mechanism for the electron transfer to the ferric ion, either free or strongly coordinated. The formation of a ferrous radical chelate can result from the dismutation reactions only when the amount of ferric ion initially present is not sufficient for complete oxidation.
Cadmium↗
Physicochemical properties of flavodoxin from Desulfovibrio vulgaris.
Reductive titration curves of flavodoxin from Desulfovibrio vulgaris displayed two one-electron steps. The redox potential E-2 for the couple oxidized flavodoxin/flavodoxin semiquinone was determined by direct titration with dithionite. E-2 was -149 plus or minus 3 mV (pH 7.78, 25 degrees C). The redox potential E-1 for the couple flavodoxin semiquinone/fully reduced flavodoxin was deduced from the equilibrium concentration of these species in the presence of hydrogenase and H-2. E-1 was -438 plus or minus 8 mV (pH 7.78, 25 degrees C). Light-absorption and fluorescence spectra of flavodoxin in its three redox states have been recorded. Both the rate and extent of reduction of flavodoxin semiguinone with dithionite were found to depend on pH. An equilibrium between the semiquinone and hydroquinone forms occurred at pH values close to the neutrality, even in the presence of a large excess of dithionite, suggesting an ionization in fully reduced flavodoxin with a pK-a = 6.6. The association constants K for the three FMN redox forms with the apoprotein were deduced from the value of K (K = 8 times 10-7 M-1) measured with oxidized EMN at pH 7.0. Oxidized flavodoxin was found to comproportionate with the fully reduced protein (k-comp = 4.3 times 10-3 M-1 times s-1, pH 9.0, 22 degrees C) and with reduced free FMN (K-comp = 44 M-1 times s-1, pH 8.1, 20 degrees C). Fast oxidation of reduced flavodoxin occurred in the presence of O-2. Slower oxidation of semiquinone was dependent on pH in a drastic way.
Desulfovibrio↗
The seven-stranded beta-barrel structure of apo-neocarzinostatin as compared to the immunoglobulin domain.
The three-dimensional structure of apo-NCS, as revealed by proton NMR, is based on an antiparallel seven-stranded beta-barrel. This fold is frequently encountered in protein structures, especially for immunoglobulin domains. The strands forming the barrel are joined by flexible loops of which three are implicated in the ligand binding site of these proteins. In this paper a preliminary comparison is given with respect to the static and dynamic properties of both the constant beta-barrel and the active loops for apo-NCS and the variable VH domain of an immunoglobulin Fab' fragment.
Amino Acid Sequence↗
Visualization of the parietal mass transfer by using immobilized peroxidase.
A new method is developed for direct visualization of the local mass transfer at solid liquid interfaces involving the chemiluminescent oxidation of luminol by H2O2 catalyzed by immobilized peroxidase. At low concentration of H2O2 (C less than 5 x 10(-5) M) this reaction is controlled by diffusion and it is possible to characterize the diffusion convection of H2O2 at each point of the tube as a function of the local hydrodynamic properties. These properties are characterized by pulsed Doppler ultrasound velocimetry. 1) The rate of light emission depends on Re1/3 for the laminar case and decreases when going downstream in accordance with the known theories of diffusion convection along tubes. 2)Downstream to a stenosis, a maximum of light appears which depends on the input Reynolds number in a manner similar to the reattachment point of the flow. This constitutes the first experimental confirmation of calculations on diffusion convection downstream to stenoses. These first experiments show the capacity of the method to detect the local properties of the parietal mass transfer phenomenon as a function of the geometry of the wall and the hydrodynamic properties of the flow.
Blood Flow Velocity↗
Gamma-radiolysis study of the reductive activation of neocarzinostatin by the carboxyl radical.
The activation of the antitumor protein antibiotic neocarzinostatin (NCS) by the carboxyl radical CO-2, a one-electron donor obtained selectively from gamma-ray irradiation of nitrous oxide-saturated formate buffer, has been investigated in the presence and in the absence of DNA at pH 4.7 and pH 7.0. The reaction of NCS with CO-2 in the absence of DNA is followed by a marked red shift (420----441 nm) and a pronounced increase (X 8.8) of the fluorescence emission corresponding to the naphthalene moiety of the NCS chromophore. The light absorption spectrum shows in parallel a hypochromic change with considerable fine structure throughout the 250-400 nm wavelength range. When DNA is present, the fluorescence intensity at completion of the reaction is slightly reduced (by 5 to 15 per cent) and the maximum emission wavelength shifted to 436-438 nm. However, the bulk rate of reaction is not altered by DNA and is independent of the pH, of the temperature and of the concentration of NCS. The NCS concentration-independence of the reaction rate is consistent with a high intrinsic rate (k greater than 10(8)M-1 . s-1) for the reaction of CO-2 with the NCS chromophore. Complete reduction of the NCS chromophore involves a total of three electron-equivalents. The final product does not react with oxygen, shows no odd electron spin, and is unable to induce DNA strand scission. Its molecular state, however, is fundamentally different when gamma-ray irradiations are performed with DNA. This bears evidence of short-lived one electron or two-electrons reduced intermediates decaying via non-identical routes depending on the presence of the acceptor DNA. Actually, dose-related strand breaks appear in DNA exposed to the action of NCS and CO-2. Some NCS chromophore-DNA covalent adducts are also found. DNA strand breakage by CO-2-activated NCS is correlated with thymine release and is inhibited by a redox-stable intercalating agent. The DNA-nicking process thus bears resemblance to that reported by other authors using mercaptans to initiate reductive activation of the NCS chromophore. However, some spectral differences are observed between the CO-2-reacted and the thiol-treated chromophores. Moreover, thymine release and strand scission in DNA incubated with CO-2 and NCS proceed under anaerobic conditions. It is proposed that the strict oxygen requirement for DNA damage by NCS in the presence of mercaptans is due, at least partly, to competition between oxygen and thiols for reaction with the same primary deoxyribose radical resulting from DNA attack by the reductively activated NCS chromophore.
Animals↗
[Regulation of cell cycle and radiation-induced cell death].
Tight control of cell proliferation is mandatory to prevent cancer formation as well as to normal organ development and homeostasis. This occurs through checkpoints that operate in both time and space and are involved in the control of numerous pathways including DNA replication and transcription, cell cycle progression, signal transduction and differentiation. Moreover, evidence has accumulated to show that apoptosis is tightly connected with the regulation of cell cycle progression. In this paper we describe the main pathways that determine checkpoints in the cell cycle and apoptosis. It is also recalled that in solid tumors radiation-induced cell death occurs most frequently through non-apoptotic mechanisms involving oncosis, and mitotic or delayed cell death.
Animals↗
[Clinical aspects of research in radiobiology. Past and future directions].
Over the last ten years the impact of fundamental radiation biology into daily radiotherapy has been of concern chiefly to fractionation, prediction of radiation response, tumour oxygenation, intrinsic radiosensitivity including genetic approaches, and the determinants of the outcome of chemoradiotherapy combinations. Future goals will rely on sophisticated approaches, based on the progress of molecular and cellular biology and the characterisation of new targets for radiation. Some of these novel advances will be discussed.
Cell Survival↗
[DNA-dependent protein kinase (DNA-PK), a key enzyme in the re-ligation of double-stranded DNA breaks].
Repair pathways of DNA are now better defined, and some important findings have been discovered in the last few years. DNA non-homologous end-joining (NEHJ) is a crucial process in the repair of radiation-induced double-strand breaks (DSBs). NHEJ implies at least three steps: the DNA free-ends must get closer, preparation of the free-ends by exonucleases and then a transient hybridisation in a region of DNA with weak homology. DNA-dependent protein kinase (DNA-PK) is the key enzyme in this process. DNA-PK is a nuclear serine/threonine kinase that comprises three components: a catlytic subunit (DNA-PKCS) and two regulatory subunits, DNA-binding proteins, Ku80 and Ku70. The severe combined immunodeficient (scid) mice are deficient in DNA-PKCS: this protein is involved both in DNA repair and in the V(D)J recombination of immunoglobulin and T-cell receptor genes. It is a protein-kinase of the P13-kinase family and which can phosphorylates Ku proteins, p53 and probably some other proteins still unknown. DNA-PK is an important actor of DSBs repair (induced by ionising radiations or by drugs like etoposide), but obviously it is not the only mechanism existing in the cell for this function. Some others, like homologous recombination, seem also to have a great importance for cell survival.
Androstadienes↗