Selective Oxidation of 2′-Deoxyguanosine to Imidazolone by the Chemical Nuclease MnTMPyP Associated to KHSO5 or Sulfite/O2.

Abstract

Mn TMPyP in the presence of sulfite/O₂ catalyses the oxidation of dG into dIz as selectively but slower and less efficiently than in the presence of KHSO_5.     

Demonstration of the O-demethylation reaction catalyzed by a peroxidase: application to 9-methoxyellipticine

9-methoxy ellipticine, an antitumor compound, is O-demethylated in presence of the system peroxidase-H2O2; this reaction yields the corresponding electrophilic quinone-imine and methanol. This O-demethylation reaction is reported for the first time and might be possibly extended to some other antitumor drugs.     

On the chemical nature of DNA and RNA modification by a hemin model system.

In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pH and fructose 2,6-bisphosphate on oxidized and reduced spinach chloroplastic fructose-1,6-bisphosphatase

This report describes the effects of pH and fructose 2,6-bisphosphate (an analog of fructose 1,6-bisphosphate) on the activity of oxidized and reduced fructose-1,6-bisphosphatase from spinach chloroplasts. Studies were carried out with either fructose 1,6-bisphosphate, the usual substrate, or sedoheptulose 1,7-bisphosphate, an alternative substrate. The reduction of the oxidized enzyme is achieved by a thiol/disulfide interchange. The pK values relative to each redox form for the same substrate (either fructose 1,6-bisphosphate or sedoheptulose 1,7-bisphosphate) are identical, suggesting the same site for both substrates on the active molecule. The finding that the analog (fructose 2,6-bisphosphate) behaves like a competitive inhibitor for both substrates also favours this hypothesis. The inhibitory effect of this sugar is more important when the enzyme is reduced than when it is oxidized. The shift in the optimum pH observed when [Mg2+] was raised is interpreted as a conformational change of oxidized enzyme demonstrated by a change in fluorescence. The reduced and oxidized forms have the same theoretical rates relative to both substrates, but the reduced form has an observed Vmax which is 60% of the theoretical Vmax while that of the oxidized form is only 37% of the theoretical Vmax. The reduced enzyme appears more efficient than the oxidized one in catalysis.     

The kinetics of the thermal deactivation of transglutaminase from guinea-pig liver

By incubating native (N) transglutaminase from guinea-pig liver at various temperatures and assaying it at 25 degrees C, two steps in the irreversible deactivation process to the denatured form (D) have been found. The fitting of the data to the equations of two possible models (the two-steps model and the two-isoenzymes model) is only compatible with the first one (N----X----D). It is shown that the structure of the active intermediate, X, depends on the deactivation temperature and on the thermal history of the enzyme. This may mean that transglutaminase exists in a large number of microstates. Surprisingly, the activation energy of deactivation is lower than that of activity (36.6 +/- 3.4 against 47.2 +/- 2.2 kJ.mol-1). By deactivating transglutaminase at a constant temperature (55 degrees C) and assaying it at variable temperatures, the activation energy of the intermediate, (X55), has been determined to be 40.2 +/- 5 kJ.mol-1, of the same order of magnitude as the native form. Among several agents assayed, only Ca2+ had a positive effect on the thermal stability of this enzyme. At 40 degrees C, transglutaminase was quite stable in the presence of Ca2+ (in its absence, the half-life was 65 min) and at 45 degrees C, its thermostability had been considerably increased, the half-life being raised from 47 min to 275 min.     

Pituitary and ovarian responses to luteinizing-hormone-releasing hormone during pregnancy and after parturition in brown hares (Lepus europaeus)

Female hares were given an i.v. injection of 5 micrograms luteinizing-hormone-releasing hormone (LHRH) between Days 7 and 19 (n = 21), 20 and 33 (n = 17) and 34 and 41 (n = 17) of pregnancy, and in the 3 days after parturition (n = 16). Whatever the stage of pregnancy, the LHRH injection induced a release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and an acute secretion of progesterone; these hormonal responses increased significantly during pregnancy, to reach values similar to those observed in nonpregnant, nonpseudopregnant females during the breeding season in the 3 days after parturition. However, the release of LH remained monophasic in pregnant and post-partum females, in contrast to the unmated females during the reproductive season, in which there was a biphasic profile. The proportion of ovulating females after LHRH treatment was approximately 60% at the beginning and end of pregnancy; and, after parturition, fell to 23% between Days 20 and 33. After Day 33, the pituitary response to LHRH was significantly higher in ovulating than in nonovulating females. At the beginning of pregnancy, 67% of females aborted after LHRH injection; after Day 20, the incidence of abortion decreased significantly and was 0% from Day 34. The amplitude and duration of progesterone secretion by the new corpora lutea resulting from ovulation after LHRH injection were similar to those of corpora lutea induced in nonpregnant females during the breeding season.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improvement of four sigma analysis for the investigation of oxygen evolution by Photosystem II

We present here an improvement to the analysis of oxygen evolution with four sigma coefficients (4-S) by computing z, the sum of the S-state probabilities, which was introduced earlier (Delrieu and Rosengard 1987, Biochim Biophys Acta 892: 163–171). We demonstrate that z is equal to the ratio of two consecutive Mean Y (the estimation of the steady state oxygen production based on local properties) found by three sigma analysis. The quantity z is useful for computing double-hits, and for showing the inactivation/activation processes of PS II complexes. Three sigma analysis assumes z=1 exactly; since this is not verified, it is argued that four sigma analysis is closer to the real workings of the water oxidizing complex. Oxygen evolution can then be interpreted in the frame of a modified Kok's model where the sum of the probabilities equals z. We therefore suggest that the closer fitting of four sigma analysis to oxygen production data is not simply due to an extra, unnecessary variable, but to the fact that PS II complexes can be inactivated and reactivated under flashing light. Finally, in order to facilitate the use of four sigma analysis, a computer program is made available upon request.     

Seasonal variations in the pituitary response to LHRH in the brown hare (Lepus europaeus)

In the brown hare, fertile mating takes place from the beginning of December to September. Pituitary and ovarian response to a monthly i.v. injection of 5 micrograms LHRH was studied from September 1983 to October 1984 in 2 groups of 6 hares. The basal concentrations of LH remained undetectable until the end of January, rose from 0.23 +/- 0.14 ng/ml from February to a maximum of 1.44 +/- 0.57 ng/ml in July. LHRH injection was always followed by a release of LH. Between September and December, the LH value peaked 15 min after injection and returned to basal concentrations 2 h later. From January, this pattern altered and a second peak of LH appeared 2 h after injection. Peak levels 15 min after LHRH were around 10 ng/ml between September and December, increased from 47.0 +/- 8.0 ng/ml in January to 106 +/- 33 ng/ml in July and decreased in August (69.4 +/- 10.6 ng/ml). The values of the second peak rose from 11.0 +/- 2.2 ng/ml in January to 90.6 +/- 12.4 ng/ml between March and July and decreased in August (24.5 +/- 5.1 ng/ml). The LH surge induced by LHRH was always followed by a transient rise in progesterone. During the breeding season, this progesterone secretion increased considerably. Ovulation was possible between January and August and the number of ovulating females was maximum between March and July. The amount and duration of progesterone secretion during the resulting pseudopregnancies increased during the breeding season.     

Peroxidase-catalyzed O-demethylation reactions. Quinone-imine formation from 9-methoxyellipticine derivatives

Despite numerous reports on the N-demethylation reactions catalyzed by peroxidases, to our knowledge, O-demethylation reactions with the same enzymes seem to be still a questionable matter. Unexpectedly, a peroxidase system (horseradish peroxidase and hydrogen peroxide) is able to effect the O-demethylation of the cytotoxic agents 9-methoxyellipticine and N2-methyl-9-methoxyellipticinium acetate. The reaction leads directly to the formation of the corresponding quinone-imine derivatives with the concomitant formation of one molecule of methanol per molecule of methoxy compound. One hydrogen peroxide molecule is consumed during the process. Experiments in H218O-enriched water clearly indicate that 18O is nearly quantitatively incorporated in the carbonyl group of the generated quinone-imine compound with the concomitant elimination of the methoxy group as methanol. So this peroxidase-catalyzed apparent O-demethylation in fact implies an oxidative demethoxylation step. This enzymatic reaction exhibits normal Michaelis-Menten saturation kinetics. Like the 9-hydroxylated ellipticines, both the 9-methoxylated ellipticines show a good affinity for the peroxidase itself (Km approximately 10 microM) but are slowly transformed to the corresponding quinone-imines. The Vmax values for methoxylated ellipticines are 10(-1)-10(-3) lower than those for hydroxylated compounds. This new route for the in vitro formation of electrophilic derivatives from the cytotoxic 9-methoxyellipticine and N2-methyl-9-methoxyellipticinium might be considered as a novel possible metabolic pathway for these drugs, especially if we bear in mind the "bio-oxidative alkylation" process previously described for at least one of the corresponding hydroxylated ellipticine derivatives (see Bernadou, J., Meunier, B., Meunier, G., Auclair, C., and Paoletti, C. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1297-1301; and Monsarrat, B., Maftouh, M., Meunier, G., Dugué, B., Bernadou, J., Armand, J. P., Picard-Fraire, C., Meunier, B., and Paoletti, C. (1983) Biochem. Pharmacol. 32, 3887-3890).     

On the mechanical characterization of compact bone structure using the homogenization theory

In a previous paper (Crolet et al., 1993, J. Biomechanics 26, 677–687), a modelling of the mechanical behavior of compact bone was presented, in which the homogenization theory was the basic tool of computation. In this simulation, approximations were used for the modelling of the lamellae and the osteons: the lamella and the osteon were divided into cylindrical sectors, each sector being approximated as a parallelepiped having a periodic structure (fibrous composite for the lamella, superimposition of plates for the osteon). The present study deals with a new model without these approximations. First, it can be proved that the homogenized elasticity tensor for a lamella, which has a non-periodic structure, is obtained at each geometrical point as a homogenized tensor of a periodic problem. Similarly, for the osteonal structure, the components of the homogenized tensor are determined at each point as the result of a periodic homogenization.

The software OSTEON, which is the computational method associated with this model, allows one to obtain a better understanding of the effects of many bony parameters. The obtained results are in accordance with experimental data.