ABSTRACT
Evidence of an overload of reactive oxygen species and peroxynitrite, a derivative of nitric oxide, in sporadic amyotrophic lateral sclerosis suggests that peroxynitrite could impair cholinergic functions. Because of the impossibility of obtaining synaptosomes from vertebrate neuromuscular junctions, we used cholinergic synaptosomes purified from Torpedo marmorata electroneurons to characterize the defects triggered by peroxynitrite in more detail. Addition of peroxynitrite or its donor 3-morpholinosydnonimine abolished high-affinity choline uptake and synthesis of acetylcholine from acetate. T. marmorata choline acetyltransferase (ChAT) was impaired to the same extent as bovine brain ChAT. A hallmark of peroxynitrite action is the nitration of tyrosine residues in proteins. Peroxynitrite induced a concentration-dependent appearance of nitrotyrosines in several neuronal proteins from synaptosomes and, more readily, from synaptic vesicles. Peroxynitrite also triggered tyrosine nitrations in purified ChAT. Peroxynitrite-dependent nitrations were impaired when synaptosomes were pretreated with thioreductants (glutathione, N-acetyl cysteine, dithiothreitol) or antioxidants (uric acid, melatonin, bovine serum albumin, desferrioxamine). Deleterious effects of peroxynitrite on choline transport and ChAT activity were prevented by the thioreductants but only partially by the antioxidants, suggesting a mechanism other than tyrosine nitration, which may involve cysteine oxidation. Further development of protective agents acting on choline transport and on ChAT activity may offer interesting therapeutic possibilities with respect to cholinergic dysfunction occurring in neurodegenerative diseases.
Subject(s)
Acetylcholine/antagonists & inhibitors , Antioxidants/pharmacology , Choline O-Acetyltransferase/metabolism , Nitrates/pharmacology , Oxidants/pharmacology , Tyrosine/analogs & derivatives , Tyrosine/metabolism , Acetates/metabolism , Acetylcholine/biosynthesis , Animals , Biological Transport/drug effects , Carbon Radioisotopes , Choline/metabolism , Choline O-Acetyltransferase/antagonists & inhibitors , Drug Interactions , Molsidomine/analogs & derivatives , Molsidomine/pharmacology , Reducing Agents/pharmacology , Torpedo , Uric Acid/pharmacologyABSTRACT
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.
Subject(s)
Glutathione/analogs & derivatives , Nitrates/metabolism , Nitric Oxide Donors/pharmacology , Nitric Oxide/metabolism , Serotonin/metabolism , Aerobiosis , Chromatography, High Pressure Liquid , Copper/pharmacology , Glutathione/pharmacology , Molsidomine/analogs & derivatives , Molsidomine/pharmacology , Nitro Compounds/analysis , Nitroprusside/pharmacology , Nitroso Compounds/analysis , Nitroso Compounds/pharmacology , Oxidants/pharmacology , Oxidation-Reduction , S-Nitrosoglutathione , Serotonin/analysis , Tyrosine/metabolismABSTRACT
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.
Subject(s)
Mammals/physiology , Nitric Oxide/physiology , Animals , Humans , Nitrates/metabolism , Protein Processing, Post-Translational , Superoxides/metabolism , Tyrosine/metabolismABSTRACT
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.
Subject(s)
Melatonin/analogs & derivatives , Melatonin/chemistry , Nitroso Compounds/chemistry , Nitrous Oxide/chemistry , Chromatography, High Pressure Liquid , Hydrogen-Ion Concentration , Reactive Nitrogen Species/chemistry , Spectrophotometry, Ultraviolet , Time FactorsABSTRACT
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.
Subject(s)
Antioxidants/chemistry , Melatonin/chemistry , Nitrates/chemistry , Nitric Oxide/chemistry , Chromatography, High Pressure Liquid , Nitrosation , Oxidation-ReductionABSTRACT
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.
Subject(s)
Nitrates/chemistry , Nitrogen Oxides/chemistry , Oxidants/chemistry , Propofol/chemistry , Anesthetics, Intravenous/chemistry , Bicarbonates/chemistry , Biotransformation , Chromatography, High Pressure Liquid , Mass Spectrometry , Nitric Oxide/chemistryABSTRACT
To react with peptides, nitric oxide.NO has to be activated by oxidation, or by coupling with superoxide (O.-2) thereby producing peroxynitrite. In the course of.NO oxidation,.NO2 free radicals and N2O3 may be formed. Using gamma-irradiation methods, we characterized the products formed by these nitrogen oxides with angiotensin II. Angiotensin II is specifically nitrated at its tyrosinyl residue by.NO2 or peroxynitrite. Equimolecular amounts of each reagent in K+/Pi solutions at pH 7.4 led to 56% and 5% nitration yields, respectively. Nitrogen oxides produced by autoxidation of.NO, as well as.NO2 under.NO, reacted only with the arginine residue, giving a mixture of peptides containing citrulline, a N-(hydroxylamino-cyanamido-) instead of guanido group, and a conjugated diene derived from an arginine side-chain. However, nitrosation reactions by N2O3 occurred only when the initial concentration of.NO2 was 10 times that able to react with angiotensin II. Thus, in this case.NO appears to protect against.NO2 action.
Subject(s)
Angiotensin II/metabolism , Nitrates/metabolism , Nitric Oxide/metabolism , Nitrogen Dioxide/metabolism , Angiotensin II/chemistry , Chromatography, High Pressure Liquid , Free Radicals/chemistry , Free Radicals/metabolism , Humans , In Vitro Techniques , Mass Spectrometry , Nitrates/chemistry , Nitric Oxide/chemistry , Nitrogen Dioxide/chemistryABSTRACT
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.
Subject(s)
Aplysia/physiology , Neurotransmitter Agents/metabolism , Neurotransmitter Agents/physiology , Nitric Oxide/pharmacology , Serotonin/metabolism , Serotonin/physiology , Acetylcholine/metabolism , Animals , Chromatography, High Pressure Liquid , Electric Stimulation , Electrophysiology , Ganglia, Invertebrate/cytology , Ganglia, Invertebrate/drug effects , Ganglia, Invertebrate/physiology , In Vitro Techniques , Membrane Potentials/physiology , Parasympathetic Nervous System/drug effects , Parasympathetic Nervous System/physiology , Patch-Clamp Techniques , Serotonin/analogs & derivatives , Synapses/drug effects , Synapses/physiology , Synaptic Transmission/drug effects , Synaptic Transmission/physiologyABSTRACT
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.
Subject(s)
Nitrates/physiology , Amino Acids/metabolism , Animals , Antioxidants/metabolism , Bicarbonates/metabolism , Carbon Dioxide/metabolism , DNA/metabolism , DNA Damage , Hemeproteins/metabolism , Humans , Hydroxylation , Nitric Oxide/metabolism , Oxidation-Reduction , Porphyrins/metabolism , Sulfhydryl Compounds/metabolism , Superoxides/metabolismABSTRACT
Nitric oxide (NO) and angiotensin II are natural regulators of blood pressure. Under aerobic conditions, NO is transformed into its higher oxides (N2O4, NO2, NO/NO2 or N2O3) and oxoperoxonitrate (currently named peroxynitrite) by coupling with superoxide. Previous studies have shown that these reactive nitrogen species should be involved in vivo in the transformation of cysteine and tyrosine into the corresponding nitrosothiol and 3-nitrotyrosine. In the present study, attention has been focused on the relative reactivities of HNO2, peroxynitrite, and NO in the presence of dioxygen, towards the arginine and tyrosine residues of the peptide angiotensin II. Nitration of the tyrosine residue is clearly the main reaction with peroxynitrite. By contrast, besides 20% of nitration of the tyrosine residue, NO in the presence of dioxygen leads to nitrosation reactions with the arginine residue similar to those observed with HNO2 at pH 5, possibly through the intermediate N2O3 reactive species. Angiotensin II is converted for the most part to peptides having lost either a terminal amine function or the whole guanido group, leading respectively to citrulline-containing angiotensin II or to a diene derivative. Identification established mainly by tandem mass spectrometry of peptidic by-products allows us to propose a cascade of nitrosations of all the amine functions of the arginine residue. Further in vivo studies show that transformations of the arginine residue in angiotensin II do not alter its vasoconstrictive properties, whereas nitration of the tyrosine residue totally inhibits them.
Subject(s)
Angiotensin II/analogs & derivatives , Angiotensin II/chemistry , Vasoconstrictor Agents/chemistry , Angiotensin II/pharmacology , Animals , Arginine/chemistry , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Structure , Molecular Weight , Nitrates/pharmacology , Nitrogen Oxides/pharmacology , Oxidants/pharmacology , Rats , Rats, Sprague-Dawley , Vasoconstriction/drug effects , Vasoconstrictor Agents/pharmacologyABSTRACT
Recent reports proposed that nitric oxide was a modulator of cholinergic transmission. Here, we examined the role of NO on cholinergic metabolism in a model of the peripheral cholinergic nervous synapse: synaptosomes from Torpedo electric organ. The presence of NO synthase was immunodetected in the cell bodies, in the nerve ending area of nerve-electroplate tissue and in the electroplates. Exogenous source of NO was provided from SIN1, a donor of NO and O2-., and an end-derivative peroxynitrite (ONOO-). SIN1 increased calcium-dependent acetylcholine (ACh) release induced by KCl depolarization or a calcium ionophore A23187. The formation of ONOO- was continuously followed by a new chemiluminescent assay. The addition of superoxide dismutase, that decreases the formation of ONOO-, did not impair the stimulation of ACh release, suggesting that NO itself was the main stimulating agent. When the endogenous source of NO was blocked by proadifen, an inhibitor of cytochrome P450 activity of NO synthase, both KCl- and A23187-induced ACh release were abolished; nevertheless, the inhibitor Ng-monomethyl-L-arginine did not modify ACh release when applied in a short time duration of action. Both NO synthase inhibitors reduced the synthesis of ACh from the radioactive precursor acetate and its incorporation into synaptic vesicles as did ONOO- chemically synthesized or formed from SIN1. In addition, choline acetyltransferase activity was strongly inhibited by ONOO- and SIN1 but not by the NO donors SNAP and SNP or, by NO synthase inhibitors. Altogether these results indicate that NO and ONOO modulate presynaptic cholinergic metabolism in the micromolar range, NO (up to 100 microM) being a stimulating agent of ACh release and ONOO- being an inhibitor of ACh synthesis and choline acetyltransferase activity.
Subject(s)
Acetylcholine/biosynthesis , Acetylcholine/metabolism , Choline O-Acetyltransferase/metabolism , Nitrates/pharmacology , Nitric Oxide/pharmacology , Synaptosomes/drug effects , Animals , Calcimycin/pharmacology , Cell Compartmentation , Electric Organ/drug effects , Electric Organ/enzymology , Electric Organ/metabolism , Enzyme Inhibitors/pharmacology , Molsidomine/analogs & derivatives , Molsidomine/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Potassium Chloride/pharmacology , Synaptosomes/enzymology , Synaptosomes/metabolism , TorpedoABSTRACT
The reactivity of catecholamines with nitrogen oxides formed from NO in aerated solutions, nitrite, and peroxynitrite was evaluated. Dopamine and norepinephrine in aerobic buffer (pH 7.4) were almost completely converted to their 6-nitro-derivatives by nitric oxide (NO) at room temperature, while epinephrine was nitrated and above all oxidized. The products obtained from each catecholamine treated with sodium nitrite at pH 4-7 were compared to those produced by NO at pH 7.4. Peroxynitrite, which can nitrate tyrosinyl residues, did not produce nitro-derivatives, only oxidized ones. The physiological relevance, particularly for the vascular and nervous system, is discussed. Catecholamine oxidation reactions could be relevant to physiological conditions and also explain neurotoxicity in Parkinson's disease and aging. Nitration reactions, requiring such high NO concentrations, do not seem possible to occur directly under normal physiological conditions, but could take place in acidic vesicules where nitrite, catecholamines, and their nitrated products could accumulate. Finally, the ability of dopamine to increase 2',5'-cyclic adenosine monophosphate (cAMP) formation in cultured striatal neurons was blocked by its nitration by NO or its nitrogen oxide derivatives.
Subject(s)
Dopamine/chemistry , Epinephrine/chemistry , Nitric Oxide/chemistry , Nitrogen Oxides/chemistry , Animals , Cells, Cultured , Corpus Striatum/cytology , Corpus Striatum/metabolism , Cyclic AMP/biosynthesis , Dopamine/metabolism , Epinephrine/metabolism , Mice , Neurons/metabolism , Nitric Oxide/metabolism , Oxidation-ReductionABSTRACT
Because NO is not very reactive in an oxygen-free buffer, a significant part of serotonin (5-HT) is transformed by NO in nondeaerated phosphate buffer, at pH 7.4, into (4-serotonyl)-4-serotonin, 4-nitrososerotonin, and 4-nitroserotonin. Dimerization and above all nitrosation occur through the HNO2 reaction in the pH 4-6 range, possibly via radical mechanism involving N2O3. 5-HT is readily a substrate for nitrosation by HNO2 or N2O3, whereas tyrosine was described as not very reactive under the same conditions. Peroxynitrite converts 5-HT to the (4-serotonyl)-4-serotonin and to the 4-nitro derivative. In order to evaluate whether such structural modifications could modulate the biological properties of 5-HT, arterial pressure was measured after i.v. bolus injection of these derivatives to anesthetized rats. Injections of the 4-nitroso- and 4-nitro-5-HT resulted in first a brief hypotensive response and did not give the subsequent hypertensive and hypotensive phases observed with 5-HT. Finally, when tested on some cloned rat 5-HT receptors stably transfected into LMTK- cells, both 4-nitroso and 4-nitro derivatives behaved as agonists and antagonists toward 5-HT1B and 5-HT2B receptors, respectively.
Subject(s)
Nitric Oxide/metabolism , Nitrogen Oxides/metabolism , Serotonin/chemistry , Animals , Blood Pressure/drug effects , Cell Line , Dimerization , Free Radicals/metabolism , Gamma Rays , Heart Rate/drug effects , Hemodynamics/drug effects , Hydrogen-Ion Concentration , Kinetics , Molecular Structure , Nitrates/metabolism , Nitro Compounds/pharmacology , Nitroso Compounds/pharmacology , Nitrous Acid/metabolism , Oxygen/metabolism , Rats , Receptors, Serotonin/metabolism , Serotonin/analogs & derivatives , Serotonin/pharmacology , Transfection/geneticsABSTRACT
The effects of Tween 20 as permeabilizing agent on tropane alkaloids fromDatura innoxia Mill. hairy root cultures have been studied. The kinetics of the alkaloid release is detailed and shows three different stages: an initial rapid increase of the alkaloid level within the roots and in the culture medium, followed by a slower but higher increase of the alkaloid concentration in the medium. During this phase, the alkaloid concentration within the roots returned to a lower value. Finally, after a longer time, the quantity of hyoscyamine in the medium decreased significantly with a variable rate. According to the total alkaloid content per flask determinations under different conditions, it is clearly demonstrated that Tween treatment permeabilized the roots, but also acted as an inducer.
ABSTRACT
Itaconate biosynthesis was studied in intact cells of high-yield (RC4') and low-yield (CM85J) strains of the fungus Aspergillus terreus by methods (tracers, nuclear magnetic resonance spectroscopy, and mass spectroscopy) that did not interfere with metabolism. Itaconate formation in RC4' required de novo protein biosynthesis. Krebs cycle intermediates increased in both strains during the production of itaconic acid. The Embden-Meyerhof-Parnas pathway and the Krebs cycle were shown to be involved in this biosynthesis by using 14C- and 13C-labelled substrates and nuclear magnetic resonance spectroscopy. A metabolic pathway for itaconate formation from glucose in A. terreus is proposed.
Subject(s)
Aspergillus/metabolism , Succinates/metabolism , Aspergillus/enzymology , Aspergillus/growth & development , Carbon Isotopes , Carbon Radioisotopes , Chromatography, High Pressure Liquid , Citric Acid Cycle , Glucose/metabolism , Magnetic Resonance Spectroscopy , Pentose Phosphate Pathway , Protein Synthesis Inhibitors/pharmacologyABSTRACT
The effects of tween 20 as permeabilizing agent on tropane alkaloids from Datura innoxia Mill, hairy roots have been studied. For various tween 20 concentrations both hyoscyamine and scopolamine accumulated in the culture medium. Plant material viability could be preserved after a 24 hours-2% tween 20 concentration treatment. The time-course study of alkaloid release showed that the maximum of excretion occurred after a 20 hour contact with tween 20. At that time, a concentration of hyoscyamine superior to 25 mg/l was detected in the medium.
ABSTRACT
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.
Subject(s)
Datura stramonium/genetics , Plants, Genetically Modified , Plants, Medicinal , Plants, Toxic , Rhizobium/genetics , Transfection/methods , Datura stramonium/embryology , Genetic Markers , Germination , Glucuronidase/genetics , Hybrid Cells , Ploidies , Seeds , Transformation, GeneticABSTRACT
Nitric oxide is synthesized in mammalian cells from L-arginine or from pharmaceutical drugs. It forms paramagnetic complexes with some metalloproteins, inhibiting key enzymes in DNA synthesis, mitochondrial respiration, iron metabolism, etc. This article reviews how electron paramagnetic resonance spectroscopy helps to detect unambiguously such specific molecular targets for NO in mammalian whole cells and organelles. EPR has also been used for the detection of spin adducts of free NO by spin-trapping methods.
Subject(s)
Nitric Oxide/metabolism , Animals , Cytochrome P-450 Enzyme System/physiology , Electron Spin Resonance Spectroscopy , Hemoglobins/metabolism , Iron/metabolism , Leukemia L1210/metabolism , Macrophages/metabolism , Nitric Oxide/chemistry , Organelles/metabolismABSTRACT
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.