Nitroglycerin metabolism by Phanerochaete chrysosporium: evidence for nitric oxide and nitrite formation.

We have demonstrated that a filamentous fungus Phanerochaete chrysosporium converts glyceryl trinitrate (GTN) into its di- and mononitrate derivatives concurrently with the formation of nitric oxide detected by electron paramagnetic resonance (EPR), and the formation of nitrite. The metabolisms of nitrite and nitrate by the fungus are evaluated and taken into account when considering GTN degradation. Lack of evidence for nitrate formation from GTN suggests that an esterase-type activity is not involved. Furthermore, the kinetics of appearance of the hemoprotein-NO and non-heme protein-NO (FeS-NO) complexes indicate that an enzymatic process producing NO directly from GTN may be involved concurrently with a glutathione transferase-like system.

Structure-activity relationships in a series of newly synthesized 1-amino-substituted ellipticine derivatives.

The synthesis of a series of 1-amino-substituted pyrido[4,3-b]carbazole derivatives, based on the substitution of corresponding 1-chloroellipticines, is reported. The cytotoxic properties on tumor cells grown in vitro, the in vivo acute toxicity of the most potent in vitro cytotoxic compounds, and the antitumor properties toward the L1210 leukemia system are described. No correlation between the apparent association constant to DNA and the in vitro cytotoxicity or the in vito antitumor efficiency could be observed in this series. 9-Hydroxylated derivatives were more cytotoxic in vitro than the corresponding 9-methoxylated compounds. However, their antitumor efficiencies on the in vivo experimental systems do not confirm the advantage of demethylation. The presence of a [(dialkylamino)alkyl]amino side chain at the 1 position of ellipticines increases the antitumor potency: 1-[[3-(diethylamino)propyl]amino]-5,11-dimethyl-6H-pyrido[4,3-b]carbazole (5) is a very potent antitumor compound (% ILS of 134 on the L1210 leukemia system).

Nitric oxide transforms serotonin into an inactive form and this affects neuromodulation.

Nitric oxide is a highly reactive molecule, diffusible and therefore ubiquitous in the central nervous system. Consequently, nitric oxide or nitric oxide-derived nitrogen oxides must enter into contact with neuromodulators and they can modify these molecules, especially monoamines, and thus change their regulatory action on synaptic transmission. We tested this possibility on a well-known, identified cholinergic synapse of Aplysia buccal ganglion, in which we have found that evoked acetylcholine release was decreased by extracellularly applied serotonin. We show that this modulatory effect of serotonin was largely reduced not only in the presence of 3-morpholinosydnonimine, a nitric oxide donor, but also when endogenous nitric oxide synthase was activated. We have shown that this decrease in the serotonin effect is due to the formation of chemical derivatives of serotonin, mainly a symmetric serotonin dimer, 4-nitroso-serotonin and 4-nitro-serotonin, which are ineffective in reproducing the modulatory effect of serotonin. Serotonin is involved in the regulation of several central functions, such as sleep-wake activity or mood. The consequences of chemical modifications of serotonin by nitric oxide must be taken into account in physiological as well as pathological situations. In addition, our results highlight the importance of the physiological implications of interactions between free radicals and neuromediators in the nervous system.

Bioconversion of glyceryl trinitrate into mononitrates by Geotrichum candidum.

Glyceryl trinitrate (GTN) 2 mM was quantitatively converted into its 1 and 2 mononitrate derivatives by Geotrichum candidum, with consumption of the nitrite ions produced. The conversion proceeded at a rate independent of the addition of either organic carbon or organic nitrogen sources. Eight batches of nitrate ester, which were added every 24 hours, were successfully converted as far as during the bioconversion process GTN concentration did not exceed 2 mM. When those limiting conditions were not observed, dramatic toxicity of GTN was noticed.

Chemical evaluation of compounds as nitric oxide or peroxynitrite donors using the reactions with serotonin.

The aim of this work was to assess the capacities of some .NO-donors to release .NO, and consequently NOx in aerobic medium, or to give peroxynitrite. The method was based on the differential reactivity of serotonin (5-HT) with either NO(x) or peroxynitrite, leading in phosphate-buffered solutions to 4-nitroso- and 4-nitro-5-HT formation, respectively. Yields and formation rates of 5-HT derivatives with .NO-donor were compared to those obtained with authentic .NO or peroxynitrite in similar conditions. Aside from the capacity of diazenium diolates (SPER/NO and DEA/NO) to release .NO spontaneously, converting 5-HT exclusively to 4-nitroso-5-HT, all other .NO donors must undergo redox reactions to produce .NO. S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP) modified 5-HT only in the presence of Cu2+, GSNO yielding 6 times more 4-nitroso-5-HT than SNP. Furthermore, in the presence of Cu+, the yield of .NO-release from GSNO was 45%. The molsidomine metabolite (SIN-1), which was presumed to release both .NO and O2(7-) at pH 7.4, reacted with 5-HT differently, depending on the presence of reductant or oxidant. Under aerobic conditions, SIN-1 acted predominantly as a 5-HT oxidant and also as a poor .NO and peroxynitrite donor (15% yield of .NO-release and 14 % yield of peroxynitrite formation). The strong oxidant Cu2+, even in the presence of air oxygen, accelerated oxidation and increased .NO release from SIN-1 up to 86%. Only a small part of SIN-1 gave simultaneously .NO and O2(7-) able to link together to give peroxynitrite, but other oxidants could enhance .NO release from SIN-1.

1-Nitrosomelatonin is a spontaneous NO-releasing compound.

Melatonin (N-acetyl-5-methoxytryptamin), the main hormone secreted by the pineal gland in mammals, is nitrosated by nitrite at acidic pH and by NO in the presence of oxygen under neutral conditions. Melatonin is also partly converted to 1-nitrosomelatonin by oxoperoxonitrate (ONOO-, peroxynitrite) in phosphate-buffered solutions at pH 7-10 [Blanchard, B., et al. (2000) Journal of Pineal Research 29, 184-192]. In the present report, we show that 1-nitrosomelatonin in turn behaves as an NO-donor regenerating melatonin. NO-release is evidenced by the formation of nitrite in phosphate-buffered solutions and oxidation of HbO2. No peroxynitrite was formed during that decomposition because serotonin used as a probe was converted only to 4-nitroso-serotonin as expected for a true NO-donor [Blanchard, B., et al. (2001) Free Radical Research, 34, 177-188]. The spontaneous decay of 1-nitrosomelatonin is not affected by GSH and metallic ions but its decomposition is accelerated in acidic pH or in the presence of NADH or ascorbate. Furthermore, melatonin is partially or entirely recovered in the absence or presence of ascorbate, respectively. A homolytic cleavage of 1-nitrosomelatonin is strongly suggested and discussed. Formation of 1-nitrosomelatonin from melatonin and reactive nitrogen species (RNS) followed by its decay into NO demonstrates that melatonin could reduce these RNS to NO.

Particular ability of cytochrome P-450 CYP3A to reduce glyceryl trinitrate in rat liver microsomes: subsequent formation of nitric oxide.

Glyceryl trinitrate was denitrated in rat hepatic subcellular fractions, with formation of glyceryl dinitrates and glyceryl mononitrates. Among differently treated-rat liver microsomes, the highest microsomal activity was obtained under anaerobic conditions with microsomal preparations from dexamethasone-treated rats and NADPH. The reaction was inhibited by O2, CO, miconazole, dihydroergotamine and troleandomycin showing that it was catalyzed by cytochrome P-450 CYP3A isoforms. The formation of a transient cytochrome P-450 Fe(II)-NO complex during this reaction was shown by visible spectroscopy. The cytosolic activity was shown to be dependent on glutathione and glutathione transferase and was not inhibited by dioxygen. In the hepatic 9000 x g supernatant containing both NADPH and cytochrome P-450 and glutathione and glutathione transferase, the cytochrome P-450-dependent reaction accounts for 30-40% of the total denitration activity observed under anaerobic conditions, using 100 microM GTN.

Intercalative binding to DNA of new antitumoral agents: dipyrido [4,3-b] [3,4-f] indoles.

The study of new intercalating DNA agents has led to the synthesis of dipyrido [4,3-b] [3,4-f] indole derivatives. These compounds bind preferentially to helical DNA and their affinity constants range from 10(6) to 10(7) M-1. The most active derivative against L1210 mouse leukemia produces viscosity changes in sheared fragments of linear DNA and in circular PM2 DNA on binding, indicating an intercalation process. The unwinding angle is estimated to be 18(0). This active derivative binds preferentially to A-T base pairs.

Antitumoral activity of dipyrido [4,3-b] [3,4-f] indoles on L 1210 leukemia.

Newly synthesized dipyrido [4,3-b] [3,4-f] indole compounds have a structural similarity with the DNA intercalating agents ellipticines (pyrido-carbazoles). The procedure of synthesis of these compounds allows the addition of a lateral chain in position 1 of the nucleus. Comparison is made between different substitutions and the resulting cytotoxic and antitumoral effects. The most active compound of the serie (1-gamma-diethyl amino propyl amino)-5-methyl-dipyrido [4,3-b] [3,4-f] indole (called here BD40) protects mice against L1210 leukemia. At the non-toxic dose of 20 mg/kg, particularly, a single infection of this compound given one day after inoculation of 10(5) L1210 cells resulted in an increased life span of 69 per cent with one survivor at 60 days. A combination of cyclophosphamide and BD40 had a synergic effect on the life span of L1210 inoculated mice. The antitumoral action of these drugs may be linked to their intercalative action upon biding to DNA.

[Hepatic steatosis in obesity in children]. / Stéatose hépatique par surcharge pondérale chez l'enfant.

The fatty liver infiltrations of the children are not uncommon and are often seen in particular diseases, mainly related to intoxication or metabolic diseases. The authors reported a case of heterogeneous fatty liver infiltration due to obesity with a good evolution after a hypocaloric diet.

Transformations of 2,6-diisopropylphenol by NO-derived nitrogen oxides, particularly peroxynitrite.

To investigate the protective effect of the anesthetic 2, 6-diisopropylphenol, or propofol, in oxidative processes in which (*)NO and peroxynitrite are involved, direct interactions were explored. The reactions of the highly lipophilic propofol with (*)NO in methanolic or aqueous buffered solutions under air were shown to produce the same compounds as those detected with peroxynitrite, but with very low yields and slow rates. In aqueous neutral medium, peroxynitrite (ONOO(-), ONOOCO(-)(2), ONOOH) was able to nitrate and oxidize propofol: In addition to oxidation products, quinone and quinone dimer, the formation of the 4-nitropropofol derivative was detected, increasing with peroxynitrite or CO(2) concentrations. Nitration reached 20% after the addition of 25 mM bicarbonate to an equimolecular mixture of peroxynitrite and propofol in methanol/phosphate-buffered solution (1/4,v/v) at pH 7.4. However, peroxynitrite either in methanol or in alkaline-buffered mixture (optimum pH 10-12) resulted in the rapid and almost complete transformation of propofol to an intermediate compound 1, which further decomposed to 4-nitrosopropofol. The transient compound 1 was obtained from either peroxynitrite or (*)NO in the presence of oxygen. From mass spectrometry determination of compound 1 we propose the involvement of the nitrosodioxyl radical ONOO(*), forming an adduct with the propofoxyl radical, to yield 4-nitrosodioxypropofol and finally 4-nitrosopropofol.

Nitrosation of melatonin by nitric oxide and peroxynitrite.

Peroxynitrite (ONOO-) is an endogenous molecule, formed by rapid coupling between *NO and O2*-. ONOO- is known to be a strong oxidant of thiols and metalloorganic compounds and also a nitrating agent of aromatic compounds such as tyrosine. However, its chemistry is not yet well elucidated under physiological conditions. Melatonin, which is an indole-amine produced by the pineal gland and other organs, has antioxidant properties. We show that melatonin reacts with ONOO- in phosphate-buffered solutions. We provide evidence of nitrosation and oxidation at the pyrrole nitrogen leading to 1-nitrosomelatonin and 1-hydroxymelatonin, these being the major reactions in aqueous phosphate-buffered solutions besides other aromatic hydroxylations and nitration. 4-Nitromelatonin is formed, but in small amounts. The kinetics of all transformations were strictly dependent on ONOO- decay, whereas yields varied with pH and the presence of CO2. The N-oxidation became competitive with nitrosation at pH 7.4, in medium containing a sufficient amount of CO2. A proposed mechanism involves the transient formation of melatonyl radical and ONOO* radical derived from ONOO- decay.

Formation of glyceryl 2-mononitrate by regioselective bioconversion of glyceryl trinitrate: efficiency of the filamentous fungus Phanerochaete chrysosporium.

Various microorganisms have been evaluated for their ability to hydrolyze glyceryl trinitrate (GTN) to glyceryl dinitrates and mononitrates. Provided that the GTN extracellular concentration was under the lethal dose, metabolite formation and regioselectivity depend on the nature of the strain used. In particular, Phanerochaete chrysosporium at a sublethal dose (3 mM) converts GTN into 1,2-glyceryl dinitrate and 2-glyceryl mononitrate (2-GMN) with a 80% regioselectivity in both steps. This bioconversion, when carried out in fermentors at 28 degrees C, allowed formation of 2-GMN at a rate of 12 mumol/h/g of dried mycelium. Successive batches of 3 mM GTN could be converted into 2-GMN as long as consecutive additions of glycerol or glucose were effected to ensure cell survival and the efficiency of the enzymatic system involved.

Effect of culture conditions on Agrobacterium-mediated transformation in datura.

A two step selection procedure is described for high frequency transformation and regeneration of transgenic plants by coculture of leaf discs of Datura innoxia with Agrobacterium tumefaciens carrying binary vectors. Leaf discs were cocultured with disarmed A. tumefaciens vectors pGS Glucl, pGSTRN943, pGV2260 and pBI121, and subcultured on regeneration media containing kanamycin. Kanamycinresistant, putatively "transformed" callus and vegetative buds were isolated, and subcultured on media containing reduced amounts of growth regulators and kanamycin to induce shooting. Rooted shoots produced normal fertile plants. Transformation frequency was related to duration of preculture, co-culture, and the bacterial strain used. With pGS Glue 1, a 3 day co-culture resulted in 70% of leaf discs being transformed. Transformation was confirmed by histochemical test for GUS activity, by the ability of leaf discs to initiate callus and from NPTII test, and Southern blot analysis. Progeny of the transgenic plants showed Mendelian segregation for kanamycin resistance.

An enzymatic method for the determination of enantiomeric composition and absolute configuration of deuterated or tritiated succinic acid.

The distribution of hydrogen isotope between pro-R and pro-S positions of succinic acid has been determined by comparison of its isotopic content before and after incubation with isocitrate lyase. This enzyme, in the presence of glyoxylate, exchanges exclusively the pro-S protons of succinate with water (M. Sprecher, R. Berger, and D. B. Sprinson (1964) J. Biol. Chem. 239, 4268-4271). With [1-14C,2(R,S)-3H]succinate as substrate, the exchange was easily followed by the decrease of 3H/14C ratio (dried aliquots), which accounted for the high isotopic effect of this reaction. The final ratio was within +/- 5% of the theoretical one. The evolution of the exchange of deuterated succinate added with [1-14C,2(R,S)-3H]succinate acid was again followed by 3H/14C ratio. The deuterium content of [2,3-2H2]succinic acid, [2-2H2]succinic acid (derived from L-[4-2H2]glutamic acid by oxidation) and of the corresponding succinates isolated after incubation with isocitrate lyase was determined by gas chromatography-mass spectroscopy of their dimethylester under NH4+ chemical ionization. This method provides the basis for a quantitative measurement of the distribution of hydrogen isotopes in unsymmetrically 2-labeled succinate or 4-labeled glutamate.

Peroxynitrite: an endogenous oxidizing and nitrating agent.

Peroxynitrite, the reaction product between nitric oxide (.NO) and superoxide, has been presumed to be a mediator of cellular and tissue injury in various pathological situations. It is formed at the convergence of two independent radical-generating metabolic pathways. Its biological effects are due to its reactivity towards a large range of molecules including amino acids such as cysteine, methionine, tyrosine and tryptophan, nucleic bases and antioxidants (e.g. phenolics, selenium- and metal-containing compounds, ascorbate and urate). Peroxynitrite reactions involve oxidation and nitration. The chemical properties depend on the presence of CO2 and metallic compounds as well as the concentrations of reagents and kinetic laws. This complex chemistry can be explained by the formation of several structural forms and active intermediates released from peroxynitrite.

[Intervention by nitric oxide, NO, and its oxide derivatives particularly in mammals]. / Intervention du monoxyde d'azote, NO, et de ses dérivés oxydés, particulièrement chez les mammifères.

Nitric oxide (NO) is a natural and stable free radical produced in soil and water by the bacteriological reduction of nitrites and nitrates and in animals by the enzyme oxidation of L-arginine. NO is biosynthesised by finely regulated enzymatic systems called NO-synthases and readily diffuses through tissues. It reacts rapidly with hemoproteins and iron-sulphur centers to form nitrosylated compounds. It oxidises more slowly to form nitrogen oxides that nitrosate thiols into thionitrite. NO is transported in these various forms and released spontaneously or through yet unclear mechanisms into most cells; it also regulates oxygen consumption at the mitochondrial respiratory chain level through interaction with cytochrome oxidase. In the cardiovascular system, NO lowers blood pressure by activating a hemoprotein, the guanylate cyclase present in muscle cells; through such interaction it acts also as a neuromediator and neuromodulator in the nervous system. However, many of NO's roles result from rapid coupling to other radicals; for example, it reacts with the superoxide anion (O2-) to form oxoperoxinitrate (ONOO-, also known as peroxynitrite). This strong oxidant of metallic centers, thiols, and antioxidants is also able to convert tyrosine to 3-nitrotyrosine and to act upon tyrosine residues contained in proteins. The biological aspects of the roles of NO are presented with particular respect to the rapid interactions of NO with hemoproteins' iron and other radicals. Concurrently, NO oxidation enables nitrosation reactions primarily of thiols but ultimately of nucleic bases. The thionitrite function (R-S-NO) thus formed and the dimerisation and nitration of tyrosine residues are protein post-translational modifications that are being investigated in animals.

Production of Agrobacterium-mediated transgenic fertile plants by direct somatic embryogenesis from immature zygotic embryos of Datura innoxia.

This work describes a new method to obtain transgenic somatic embryos from Agrobacterium-infected immature zygotic embryos of Datura innoxia. It has several advantages over previous transformation methods such as the absence of a callus phase, an average transformation rate of 76% and a high regeneration frequency. Critical steps for optimal transformation were the embryo stage and a short preculture treatment. The marker gene beta-glucuronidase and light microscopy were used to identify the competent embryogenic cells which, after transformation, passed through the classical stages of embryo development. The transgenes were transmitted to the progeny in a Mendelian fashion. The plants regenerated via direct somatic embryogenesis were cytologically and morphologically uniform. We also observed that: (1) wounding or wound-induced divisions were not required for zygotic embryo transformation; (2) epidermal cells were competent for both transformation and regeneration; and (3) competency for Agrobacterium infection was developmental stage-specific. This new method should facilitate the development of new strategies to routinely transform recalcitrant plant species.