Azide-dependent nitric oxide emission from the water fern Azolla pinnata

Nitric oxide (NO) is involved in versatile functions in plant growth and development as a signaling molecule. To date, plants have been reported to produce NO following exposure to nitrite (N O ₂ ^? ) the amino acid L-arginine, hydroxylamine, or polyamines. Here we demonstrate azide-dependent NO production in plants. The water fern Azolla pinnata emitted NO into air upon exposure to sodium azide (NaN₃). The NO production was dependent on azide concentration and was strongly inhibited by potassium cyanide (KCN). Incubation of A. pinnata with the catalase inhibitor 3-aminotriazole (3-AT) abolished the azide-dependent NO production. Although nitrite-dependent NO production was inhibited by sodium azide, azide-dependent NO production was not affected by nitrite. These results indicate that A. pinnata enzymatically produces NO using azide as a substrate. We suggest that plants are also capable of producing NO from azide by the action of catalase as previously reported in animals.     

Mutation spectrum of a binary mixture of mutagens (methapyrilene and sodium azide) in strain TA1535 of Salmonella

Methapyrilene (MP) is a rat-liver carcinogen and cocarcinogen that exhibits a narrow spectrum of mutagenic activity in Salmonella typhimurium, inducing only a 2-fold increase in revertants only in the base-substitution strain TA1535; it also enhances the mutagenic activity of sodium azide (NaN₃) in the same strain. To examine the effects of MP at the molecular level, we used the colony probe hybridization procedure dveloped by Cebula and Koch (Mutation Res., 229 (1990) 79–87) to identify the base substitutions in ~800 background, MP-, NaN₃-, and MP + NaN₃-induced revertants of the hisG46 allele of strain Ta1535. The predominant mutation in all 4 mutation spectra was a CCC → CTC transition. the results suggest a mechanism by which MP enhances the infidelity of the DNA replication complex or inhibits a DNA reapir of proofreading function, resulting in the production of more of the same error that occurs normally and that is also induced by NaN₃. Such a mechanism might be the basis for the carcinogenic and cocarcinogenic activities of MP. To our knowledge, this is the first report of the molecular analysis of mutants produced by exposure of cells to a binary mixture of mutagens.     

Involvement of Singlet Oxygen in 5-Aminolevulinic Acid-Induced Photodynamic Damage of Cucumber (Cucumis sativus L.) Chloroplasts

Cucumber (Cucumis sativus L., cv Poinsette) plants were sprayed with 20 millimolar 5-aminolevulinic acid and then incubated in the dark for 14 hours. The intact chloroplasts were isolated from the above plants in the dark and were exposed to weak light (250 micromoles per square meter per second). Within 30 minutes, photosystem II activity was reduced by 50%. The singlet oxygen (¹O₂) scavengers, histidine and sodium azide (NaN₃) significantly protected against the damage caused to photosystem II. The hydroxyl radical scavenger formate failed to protect the thylakoid membranes. The production of ¹O₂ monitored as N,N-dimethyl p-nitrosoaniline bleaching increased as a function of light exposure time of treated chloroplasts and was abolished by the ¹O₂ quencher, NaN₃. Membrane lipid peroxidation monitored as malondialdehyde production was also significantly reduced when chloroplasts were illuminated in the presence of NaN₃ and histidine. Protochlorophyllide was the most abundant pigment accumulated in intact chloroplasts isolated from 5-aminolevulinic acid-treated plants and was probably acting as type II photosensitizer.     

The Effects of Cyanide and Azide on the Photoreduction of 3-Phosphoglycerate and Oxaloacetate by Wild Type and Two Reductive Pentose Phosphate Cycle Mutants of Chlamydomonas reinhardtii

3-Phosphoglycerate- and oxaloacetate-dependent O₂ photoevolution by permeabilized cell preparations (Pressates), prepared from wild type (Wt) and two reductive pentose phosphate cycle mutants of Chlamydomonas reinhardtii showed different sensitivities to the inhibitors sodium cyanide and sodium azide. NaCN (1.5 millimolar) severely inhibits both CO₂- and 3-phosphoglycerate-dependent O₂ photoevolution by the Wt Pressate, but does not inhibit 3-phosphoglycerate-dependent O₂ photoevolution by Pressates prepared from the mutants rcl-u-1-10-6C (which lacks ribulose, 1-5, bisphosphate carboxylase activity) and F60 (which lacks phosphoribulokinase activity). NaN₃ (0.5 millimolar) inhibits 3-phosphoglycerate-dependent O₂ photoevolution by the rcl-u-1-10-6C Pressate more severely than in the Pressates prepared from F60 and Wt. A higher concentration of NaN₃ (2.0 millimolar) severely inhibited oxaloacetate-dependent O₂ photoevolution by the rcl-u-1-10-6C, but not by the F60 Pressate. O₂ exchange-dependent upon methyl viologen was not strongly inhibited by 2 millimolar NaN₃ in either of the mutant Pressates. The data suggests that the mutational lesions which resulted in decreased ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase activities effected changes in other photosynthetic reactions, either by direct interactions between component proteins or by causing changes in substrate or cofactor availability to the partial reactions.     

New coordination polymer based on salpn Schiff base and azide bridging ligands: Synthesis and structural characterization of {Na[Co(μ-salpn)(μ1,1-N3)2]}n (H2salpn = N,N′-bis(salicylidene)-1,3-diaminopropane)

One-pot reaction of cobalt(II) nitrate hexahydrate Co(NO₃)₂ · 6H₂O with H₂salpn (N,N′-bis(salicylidene)-1,3-diaminopropane) in presence of a large excess of sodium azide (NaN₃) gives the new Co(III) compound {Na[Co^III(μ-salpn)(μ_1,1-N₃)₂]}_n (1), which was characterized by single crystal X-ray diffraction analysis. The crystal structure shows polymeric 1D complex generated by the hexadentate Schiff base salpn²⁻ and two crystallographically different azide ligands. The two nitrogen atoms of the salpn ligand are bonded to the cobalt(III) ion while each phenoxo oxygen atom is bonded to the same Co(III) ion and to two equivalent sodium ions. Each azide ligand acts with the end-on bridging coordination mode between Co(III) and Na(I) ions. The Co(III) ion adopts a distorted octahedral geometry arising from two oxygen and two nitrogen atoms of the salpn ligand and from two nitrogen atoms of the two crystallographically different azide ligands in trans positions. Such [Co(salpn)(N₃)₂] entities are connected each other by sodium ions through four oxygen atoms of two equivalent Schiff base ligands and two nitrogen atom of the two different azide ligands to generate the 1D structure of 1.     

Mutagenicity of nitrofuran derivatives, including furylfuramide, a food preservative.

The effects of sodium azide (NaN₃) in combination with diethyl sulfate (dES) or N-methyl-N′-nitrosourea (MNH) on mutation frequency in barley were studied. It was found that sodium azide produced high frequencies of chlorophyll mutations when used alone and has a synergistic effect on mutation yields following MNH treatments. However, the mutation frequency was decreased whe azide was applied following dES treatment of seeds. The mutagenic efficiency of azide was found to be high, possibly because of low “physiological” damage. The synergistic increase in mutation yields by MNH and azide treatment indicates that azide has unusual promise as a mutagen for both practical and research applications.     

The glutamate receptor-like protein of brain synaptic membranes is a metalloprotein.

The glutamate binding glycoprotein from rat brain synaptic plasma membranes was found to be quite sensitive to inhibition by sodium azide (NaN₃. The glutamate binding activity of the purified glycoprotein was decreased to 50% in the presence of 0.33 mM NaN₃. Similar concentrations of NaN₃ also caused 50% inhibition of glutamate binding to the synaptic membranes. Analysis of the purified binding protein for its content of iron and sulfur revealed the presence of 1.95 g atom Fe and 2.19 g atom S per mole of 14,000 M.W. protein. On the basis of changes in the difference spectra of this protein it is believed that both the azide effect and the glutamate binding are taking place at a site which contains the Fe₂S₂ center.     

Catalase catalyzes nitrotyrosine formation from sodium azide and hydrogen peroxide

Sodium azide (NaN₃) is known as an inhibitor of catalase, and a nitric oxide (NO) donor in the presence of catalase and H₂O₂. We showed here that catalase-catalyzed oxidation of NaN₃ can generate reactive nitrogen species which contribute to tyrosine nitration in the presence of H₂O₂. The formation of free-tyrosine nitration and protein-bound tyrosine nitration by the NaN₃/catalase/H₂O₂ system showed a maximum level at pH 6.0. Free-tyrosine nitration induced by peroxynitrite was inhibited by ethanol and dimethyl-sulfoxide (DMSO), and augmented by superoxide dismutase (SOD). However, free-tyrosine nitration induced by the NaN₃/catalase/H₂O₂ system was not affected by ethanol, DMSO and SOD. NO^-₂ and NO donating agents did not affect free-tyrosine nitration by the NaN₃/catalase/H₂O₂ system. The reaction of NaN₃ with hydroxyl radical generating system showed free-tyrosine nitration, but no formation of nitrite and nitrate. The generation of nitrite (NO^-₂) and nitrate (NO^-₃) by the NaN₃/catalase/H₂O₂ system was maximal at pH 5.0. These results suggested that the oxidation of NaN₃ by the catalase/H₂O₂ system generates unknown peroxynitrite-like reactive nitrogen intermediates, which contribute to tyrosine nitration.     

Effects of Sodium Azide on Sodium Fluxes in Frog Striated Muscle

Unidirectional Na fluxes from frog''s striated muscle were measured in the presence of 0 to 5 mM sodium azide. With azide concentrations of 2 and 5 mM the Na efflux was markedly stimulated; the Na efflux with 5 mM azide was about 300 per cent greater than normal. A similar increase was present when all but the 5.0 mM sodium added with azide was replaced by choline. 10^-5 M strophanthidin abolished the azide effect on Na²⁴ efflux. Concentrations of azide of 1.0 mM or less had no effect on Na efflux. The Na influx, on the other hand, was only increased by 41 per cent in the presence of 5 mM NaN₃. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of azide. The hypothesis is advanced that the active transport of Na is controlled by the transmembrane potential and that the stimulation of Na efflux is produced as a consequence of the membrane depolarization caused by the azide.     

Responses of Arabica coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L. var. Catuaí) cell suspensions to chemically induced mutagenesis and salinity stress under <Emphasis Type="Italic">in vitro</Emphasis> culture conditions

Crop improvement of Coffea arabica L. (coffee) via mutagenesis could accelerate breeding programs; thus, the present study aimed to develop an in vitro protocol using the chemical mutagens sodium azide (NaN₃) and ethyl methanesulfonate (EMS) on embryogenic cell suspensions of Arabica coffee variety Catuaí and, subsequently, to evaluate the responses of the resulting mutagenized tissues to salinity stress. Embryogenic suspension cultures were incubated with 0.0, 2.5, 5.0, or 10.0 mM NaN₃ or 0.0, 185.2, 370.5, or 741.0 mM EMS. As the concentration of NaN₃ or EMS increased, the survival of embryogenic suspension cultures decreased compared to controls. The median lethal dose (LD₅₀) for NaN₃ was 5 mM for 15 min and for EMS it was 185.2 mM for 120 min. Embryogenic suspension cultures treated with NaN₃ or EMS were cultured on selective medium supplemented with 0, 50, 100, 150, 250, or 300 mM NaCl showed that 50 mM NaCl could be used as selection pressure. Plantlet growth and total amino acid content were affected by NaCl stress; some mutants had longer shoots and higher amino acid content than controls. Random amplified polymorphic DNA (RAPD) analysis was performed to determine whether the NaN₃ or EMS treatments could induce genetic variability and resulted in identifiable polymorphic markers. A total of 18 10-mer primers were used to amplify genomic DNA of putative mutant and non-mutant arabica coffee embryogenic cultures and produced 50 scorable bands, of which 22% were polymorphic.     

Activation of rat adipocyte plasma membrane adenylate cyclase by sodium azide

The adenylate cyclase of rat adipocyte plasma membrane is stimulated by sodium azide with a half maximal activation of 100–150% occuring at 50 mM NaN₃. Studies of the effects of azide and fluoride indicate different mechanisms of stimulation of the enzyme by these ions. Comparable stimulation of the activity is obtained by 100 mM NaN₃ or 10 mM NaF but unlike azide, higher concentrations of fluoride cause inhibition of the enzyme. Fluoride activated adenylate cyclase is further stimulated by azide. Epinephrine stimulation of the enzyme is absent in the presence of fluoride but the hormone enhances the activity in the presence of azide. Reversal of the inhibitory action of GTP on adenylate cyclase by epinephrine is demonstrated even in the presence of azide but not in the presence of fluoride.     

Does endogenous cyclic GMP inhibit potassium stimulated 45Ca uptake by P2 fraction from rat brain?

The effects of sodium azide (NaN₃), hydroxylamine (NH₂OH) and sodium nitroprusside on potassium-stimulated ⁴⁵Ca uptake (K-stimulated ⁴⁵Ca uptake) by P₂ fraction of Gray and Whittaker from rat brain were investigated. During preincubation with these reagents, the contents of cyclic GMP in synaptosomes increased, reaching maximum levels within 2 min. On preincubation for 2 min, NaN₃, an activator of membrane bound guanylate cyclase, inhibited K-stimulated ⁴⁵Ca uptake, but NH₂OH and sodium nitroprusside did not affect it. Sodium cyanide, another metabolic inhibitor, had no effect on K-stimulated ⁴⁵Ca uptake. There was a correlation between inhibition of K-stimulated ⁴⁵Ca uptake and increase in the cGMP level on preincubation with NaN₃ for various periods. Based on these results role of cGMP in or around the membrane was discussed in relation to the K-stimulated ⁴⁵Ca uptake by P₂ fraction.     

Mitochondrial ERK plays a key role in delta-opioid receptor neuroprotection against acute mitochondrial dysfunction

It is well established that stimulating delta-opioid receptor (DOR) with its specific agonists elicits neuroprotection against hypoxia/ischemia. Mitochondrial dysfunction plays a key role in hypoxic neuronal injury, but the effects of DOR activation on mitochondrial dysfunction in neurons are poorly elucidated. In this investigation, we studied the effects of [d-Ala2, d-Leu5] enkephalin (DADLE), a potent DOR agonist, on acute mitochondrial dysfunction and ensuing cell damage induced by sodium azide in primary rat cortical neuronal cultures, and explored possible mechanisms underlying. Here, we show that DADLE reverses NaN₃-induced acute mitochondrial dysfunction by selectively activating DOR, mainly including mitochondrial membrane depolarization, mitochondrial Ca²⁺ overload and reactive oxygen species generation. DOR stimulation also inhibits cytochrome c release and caspase-3 activation, and attenuates neuronal death caused by acute NaN₃ insults. Furthermore, DOR activation with DADLE protects neurons from acute NaN₃ insults mainly through PKC-ERK pathway, and mitochondrial ERK activation is especially required for DOR neuroprotection against acute mitochondrial dysfunction.     

Instability of urinary excreted methyl-2-acetamido-2-deoxy-1-seleno-β-d-galactopyranoside (selenosugar 1), the main elimination product of human selenium metabolism,and measures for its stabilization

BackgroundThe urinary excreted selenium species selenosugar 1 (SeSug1) plays a key role for monitoring of supplemental selenium exposure, e.g. by occupational exposure. In order to reproduce its contents in the long term, the integrity of SeSug1 in the urine is essential. Studies on the stability of SeSug1 in urine samples stored at −20 °C have shown that degradation of SeSug 1 occurs, requiring adequate countermeasures.MethodsHere, we explored the long-term stability of SeSug1 under usual storage conditions at −20 °C. For this purpose, the simultaneous determination of selenosugar 1 and methylselenic acid (MeSeA) was used to explore the stabilizing of the SeSug1 content by applying sodium azide (NaN₃) as a bactericide or/and 5 M ammonium acetate buffer for pH control.ResultsIn untreated urine, conversion of SeSug1 to MeSeA was evident within days. Differences in urine matrices clearly showed different impact, which could be attributed to different buffer strengths by the urine itself. For durability, various concentrations of sodium azide were first applied, followed by pH buffering. A combination of 0.1% NaN₃ and pH of 5.5 kept the SeSug1 content stable for over 3 months.ConclusionThe formation of MeSeA as degradation product of SeSug1 could be confirmed. Based on the proportions, an oxidation-based decomposition pathway was proposed. The investigations revealed that the complex interaction of pH buffering and bactericidal activity must be taken into account in order to stabilize SeSug1 in the urine. The main effect was the addition of NaN₃. However, the alkaline nature of NaN₃ required a sufficient buffering of the urinary matrix at a pH of 5.5.     

Effect of vanadate on gill cilia: Switching mechanism in ciliary beat

Lateral (L) cilia of freshwater mussel (Margaritana margaritifera and Elliptio complanatus) gills can be arrested in one of two unique positions. When treated with 12.5 mM CaCl₂ and 10^?5 M A23187 they arrest in a “hands up” position, ie, pointing frontally. When treated with approximately 10 mM vanadate (V) they arrest in a “hands down” position, ie, pointing abfrontally. L-cilia treated with 12.5 mM CaCl₂ and 1 mM NaN₃ also arrest in a “hands down” position; substitution of 20 mM KC1 and 1 mM NaN₃ causes cilia to move rapidly and simultaneously to a “hands up” position. The observations suggest that there are two switching mechanisms for activation of active sliding in ciliary beat one at the end of the recovery stroke and the other at the end of the effective stroke; the first is inhibited by calcium and the second by vanadate or azide. This is consistent with a model of ciliary beating where microtubule doublet numbers 1, 2, 3, and 4 are active during the effective stroke while microtubule doublets numbers 6, 7, 8, and 9 are passive, and the converse occurs during the recovery stroke.     

Novel dextranase catalyzing cycloisomaltooligosaccharide formation and identification of catalytic amino acids and their functions using chemical rescue approach

A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7–14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr⁴⁵¹–Val¹⁰⁸²), a portion of which shares identity (35% at Ala³⁹–Ser¹³⁰⁴ of PsDex) with Pro³²–Ala⁷⁵⁵ of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val⁸³⁷–Met⁹³² for PsDex and Tyr⁴⁰⁴–Tyr⁴⁹² for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala³⁹–Ser¹³⁰⁴) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp¹⁸⁹, Asp³⁴⁰, Glu⁴¹², and Asp¹²⁵⁴ of PsDex) of catalytic candidates. Their amide mutants decreased activity (1/1, 500 to 1/40, 000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN₃. D340G or E412Q formed a β- or α-isomaltotetraosyl azide, respectively, strongly indicating Asp³⁴⁰ and Glu⁴¹² as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN₃.     

Energy pathways associated with sustained spermatozoon motility in the endangered Siberian sturgeon Acipenser baerii

Sturgeon spermatozoa are unique for their sustained motility. We investigated the relative importance of bioenergetic pathways in the energy supply of Siberian sturgeon Acipenser baerii spermatozoa during motile and immotile states. Spermatozoon motility and oxygen consumption rate (OCR) were analysed following exposure to inhibitors of oxidative phosphorylation (sodium azide, NaN₃), glycolysis (2-deoxy-D-glucose, DOG) and β-oxidation of fatty acids (sodium fluoride, NaF), and to an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenyl hydrazine, CCCP). No significant difference in curvilinear velocity was observed after addition of these reagents to activation medium (AM) or nonactivation medium (NAM) for incubation. Incubation of spermatozoa in NAM containing CCCP or NaN₃ resulted in significantly decreased motility duration compared to controls. The OCR of sturgeon spermatozoa in AM (11.9 ± 1.4 nmol O₂ min⁻¹ (10⁹ spz)⁻¹) was significantly higher than in NAM (8.2 ± 1.5 nmol O₂ min⁻¹ (10⁹ spz)⁻¹). The OCR significantly declined with addition of NaN₃ to AM and NAM. No significant difference in motility parameters or OCR was observed with NaF or DOG. These results suggest active oxidative phosphorylation in both immotile and motile spermatozoa. Nevertheless, mitochondrial respiration occurring during motility is not sufficient to meet the high energy demands, and the energy required for sustained motility of Siberian sturgeon spermatozoa is derived from adenosine triphosphate accumulated during the quiescent state.     

Mutagenesis of soybean (Glycine max (L.) Merr.) and the isolation of non-nodulating mutants

Soybean seeds (cv. Bragg) were mutagenized with either ethyl methanesulphonate (EMS), gamma-rays or sodium azide (NaN₃). EMS was the most efficient in generating chlorophyll-deficient variants in the M₂. NaN₃ was not an effective mutagen. Approximately 25 200 M₂ plants were screened for absence of nodulation after inoculation with Bradyrhizobium japonicum strain CB1809 (=USDA136). Three stable non-nodulation mutants were recovered and designated nod49, nod772 and nod139. Mutant nod49 was isolated from a mixture of M₂ populations and this mutant also failed to nodulate upon inoculation with Rhizobium fredii strain USDA257, which nodulated the parent cultivar. Mutants nod772 and nod139 came from segregating EMS-derived M₂ families, indicating that these mutations were a result of mutagenesis. Inheritance of the non-nodulation character has been demonstrated through to the M₅, M₄ and M₃ generations for nod49, nod772 and nod139, respectively.     

Synthesis and characterization of oligodeoxynucleotides containing a novel tetraazabenzo[cd]azulene:naphthyridine base pair

The synthesis and thermal stability of oligodeoxynucleotides (ODNs) containing 4-amino-2,3,5,6-tetraazabenzo[cd]azulen-7-one nucleosides 5 (BaO^N) with the aim of developing new base pairing motif is described. The tricyclic nucleoside 5 was prepared starting with the 7-deaza-7-iodopurine derivative 1 via a palladium catalyzed cross-coupling reaction with methyl acrylate, followed by an intramolecular cyclization. The resulting nucleoside was incorporated into ODNs, and the base pairing property of the BaO^N:NaN^O (2-amino-7-hydroxy-1,8-naphthyridine nucleoside) pair in the duplex was evaluated by a thermal denaturation study. The melting temperature (T_m) of the duplex containing the BaO^N:NaN^O pair showed a higher value than that of the duplexes containing the adenine:thymine (A:T) and the guanine:cytosine (G:C) pairs, however it was lower than that of the ImO^N:NaN^O (ImO^N = 7-amino-imidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidin-4(5H)-one nucleoside) pair. A temperature-dependent ¹H NMR study revealed that the H-bonding ability of BaO^N was lower than that of ImO^N, which would explain why the BaO^N:NaN^O pair was less thermally stable than the ImO^N:NaN^O pair.