Sodium Azide Treatment Research Articles

Effects of chemical ischemia in cerebral cortex slices: Focus on nitric oxide

Superfused rat cerebral cortex slices were submitted to a continuous electrical (5 Hz) stimulation and treated with sodium azide (1–10 mM) in the presence of 2 mM 2-deoxyglucose (“chemical ischemia”). Presynaptic cholinergic activity, evaluated as acetylcholine release, was inhibited depending on the sodium azide concentrations and on the length of application (5–30 min). Following a 5-min treatment with 10 mM sodium azide, acetylcholine release was reduced to 45 ± 2.3%; simultaneously, there was a 15- and 10-fold increase in glutamate and nitric oxide effluxes, respectively. After restoring normal superfusion conditions, acetylcholine release recovered to 70 ± 3.1% of the controls; the N-methyl- d-aspartate receptor antagonist MK-801 (10 μM) as well as the nitric oxide scavengers, haemoglobin (20 μM) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (150 μM), improved the recovery in presynaptic activity, showing that both glutamate and nitric oxide play detrimental roles in chemical ischemia. On the other hand, the post-ischemic recovery was worsened by the guanylylcyclase inhibitor 1H-[l,2,4]oxadiazolo[4,3,- a]quinoxalin-1-one (10 μM), suggesting that the activation of such a pathway plays a neuroprotective role and that the nitric oxide-induced harmful effects depend on different mechanisms. Chemical ischemia-evoked nitric oxide efflux partly derived from its calcium-dependent endogenous synthesis, since both the intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (1 mM), and the nitric oxide synthase inhibitor, N ω-nitro- l-arginine methyl ester (100 μM), substantially prevented sodium azide effects. Nitric oxide efflux was only weakly reduced by MK-801 and was not modified by either the L-type calcium channel blocker, nifedipine (10 μM) or the N-type calcium channel blocker ω-conotoxin (0.5 μM), thus suggesting a prevailing intracellular calcium-dependence of nitric oxide production, although a partial extracellular calcium source cannot be ruled out. These findings show that sodium azide plus 2-deoxyglucose treatment is a useful protocol to induce brain ischemia in vitro and underline the involvement of nitric oxide in the complex events following the ischemic insult.

Kinetics of peat soil dissolved organic carbon release from bed sediment to water. Part 1. Laboratory simulation

Temporary water reservoirs built upon peat soil may exhibit water quality impairment from elevated dissolved organic carbon (DOC). Microbiological decay of the organic carbon in the bed with subsequent release produces “tea” colored water which may require treatment prior to use. This paper describes laboratory experiments designed to obtain data on the release process of DOC from soils containing 0.65%, 10% and 19% carbon. Parallel experiments with and without sodium azide treatment clearly distinguished the initial release of a porewater residual fraction and the microbial produced fraction. A one to two day quick-release DOC fraction, which ranges from 28% to 50%, first emerged from the bed, Step-1. This was followed by a constant rate of DOC production over four weeks, Step-2. The Step-2 average production rates were 3.4, 12, and 31 mg DOC/kg(dry)/day for the respective soils and increased as soil carbon content increased. The inorganic carbon (IC) behaved oppositely; its rate of production decreased with increasing soil carbon. A consistent and simple rate equation described the Step-2 DOC production process. This and other evidence obtained provided the basis for developing a mathematical model that couples both steps of the bed-to-water DOC release chemodynamics. The model is presented in a companion paper (Part-2).

Cells resistant to HTI-286 do not overexpress P-glycoprotein but have reduced drug accumulation and a point mutation in α-tubulin

Abstract HTI-286, a synthetic analogue of hemiasterlin, depolymerizes microtubules and is proposed to bind at the Vinca peptide site in tubulin. It has excellent in vivo antitumor activity in human xenograft models, including tumors that express P-glycoprotein, and is in phase II clinical evaluation. To identify potential mechanisms of resistance induced by HTI-286, KB-3-1 epidermoid carcinoma cells were exposed to increasing drug concentrations. When maintained in 4.0 nmol/L HTI-286, cells had 12-fold resistance to HTI-286. Cross-resistance was observed to other Vinca peptide-binding agents, including hemiasterlin A, dolastatin-10, and vinblastine (7- to 28-fold), and DNA-damaging drugs, including Adriamycin and mitoxantrone (16- to 57-fold), but minimal resistance was seen to taxanes, epothilones, or colchicine (1- to 4-fold). Resistance to HTI-286 was retained when KB-HTI-resistant cells were grown in athymic mice. Accumulation of [3H]HTI-286 was lower in cells selected in intermediate (2.5 nmol/L) and high (4.0 nmol/L) concentrations of HTI-286 compared with parental cells, whereas accumulation of [14C]paclitaxel was unchanged. Sodium azide treatment partially reversed low HTI-286 accumulation, suggesting involvement of an ATP-dependent drug pump. KB-HTI-resistant cells did not overexpress P-glycoprotein, breast cancer resistance protein (BCRP/ABCG2/MXR), MRP1, or MRP3. No mutations were found in the major β-tubulin isoform. However, 4.0 nmol/L HTI-286-selected cells had a point mutation in α-tubulin that substitutes Ser for Ala12 near the nonexchangeable GTP-binding site of α-tubulin. KB-HTI-resistant cells removed from drug became less resistant to HTI-286, no longer had low HTI-286 accumulation, and retained the Ala12 mutation. These data suggest that HTI-286 resistance may be partially mediated by mutation of α-tubulin and by an ATP-binding cassette drug pump distinct from P-glycoprotein, ABCG2, MRP1, or MRP3.

Technological characteristics of newly developed mutant common winter wheat lines

Fifteen hybrid-mutant lines and two direct mutant lines were studied in terms to their technological quality in 2000 and 2001 vegetation years. The hybrid-mutant lines were produced using chemical mutagen sodium azide at a concentration of 1mM on F<sub>2</sub> seeds. For parent cultivars, promising and well adapted Bulgarian and foreign common winter wheat cultivars were used. The direct mutant lines were obtained by gamma-irradiation and sodium azide treatment of dry seed from cultivars. The differences of the following three characteristics: quality index, softening of dough and energy for dough deformation (W) of MX 77/14 compared to the total mean value are positive and statistically significant. Crude protein, softening of dough, and energy for dough deformation (W) of MX 84/37 are also significantly different in relation to the total mean value. Both wheat lines refer to the group of common winter wheat with very good technological quality. This fact was confirmed of higher values of energy for dough deformation (W), than strong wheat cultivar Pobeda.

Uptake of proteins and degradation of human serum albumin by Plasmodium falciparum – infected human erythrocytes

BackgroundIntraerythrocytic malaria parasites actively import obligate nutrients from serum and export proteins and lipids to erythrocyte cytoplasm and membrane. The import of macromolecules in the malaria parasite has been the subject of many debates. To understand the import of macromolecules by the parasite, we studied the uptake of proteins by Plasmodium falciparum infected human erythrocyte.MethodsProteins were biotin labelled individually, purified on a gel filtration column and added to uninfected and infected asynchronized culture. The uptake of these proteins by malaria parasites was determined by western blot analysis of parasite pellet and their different fractions using streptavidin-horseradish conjugate. To further confirm this import, we studied the uptake of 125I-labelled proteins by western blot analysis as well as used direct immunofluorescence method.ResultsHere we show that biotin labelled and radio-iodinated polypeptides of molecular sizes in the range of 45 to 206 kDa, when added in the culture medium, get direct access to the parasite membrane through a membrane network by by-passing the erythrocyte cytosol. The import of these polypeptides is ATP-dependent as sodium azide treatment blocks this uptake. We also show that malaria parasites have the ability to take up and degrade biotin labelled human serum albumin, which has been shown to be essential for the parasite growth.ConclusionsThese results can be used, as a basis to explore the role of human serum albumin in the intraerythrocytic development of parasites, and this in turn can be an important adjunct to the development of novel antimalarial drugs.

Involvement of adaptive mechanism and an energy-dependent efflux system of Pseudomonas sp. LE2 counteracting insecticide lindane

An adaptive mechanism and efflux-transport system of Pseudomonas sp. LE2 counteracting organochlorine insecticide lindane were investigated to understand the survival mechanism of this microorganism in lindane-contaminated environment. Changes in membrane fluidity and fatty acid composition of Pseudomonas sp. LE2 cells grown on glucose either in the presence or absence of lindane were determined. Considerably high ratio of total saturated to unsaturated fatty acids in cells grown on glucose in the presence of lindane was observed as compared to that in cells grown on glucose in the absence of lindane. Lindane significantly affected in vitro the membrane of Pseudomonas sp. LE2 cells by decreasing the fluorescence polarization of membrane probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) inserted into cytoplasmic membrane. Less fluidizing effect of lindane on the membranes of cells that had adapted to lindane was observed. Lindane transport was assessed by incubating cell suspensions with [ 14C]lindane either in the presence or absence of sodium azide, a membrane transport inhibitor, and by measuring the radioactivity in cell pellets. No time-dependent and steady-state increase in the radioactivity of [ 14C]lindane without sodium azide treatment was observed in cell pellets, suggesting simple diffusion mechanism for lindane accumulation in cells. Significant increase in the radioactivity was observed in cell pellets by sodium azide treatment. Sodium azide inhibited the efflux of lindane by Pseudomonas sp. LE2. These results suggest the view that Pseudomonas sp. LE2 capable of growing in the presence of lindane is able to adapt to the fluidizing action of lindane by incorporating the saturated fatty acid into membrane lipids, and eliminate lindane from cells by an energy-dependent efflux mechanism.

Sodium azide treatment decreases striatal and cortical concentrations of alpha-tocopherol in rats.

Sodium azide (20mg/kgsc), given for a maximum of 3 days to rats, significantly decreased the alpha-tocopherol concentrations in the cortex on day 2 and in the striatum on day 3. In these brain regions the oxidized glutathione values showed 30 to 36% (statistically not significant) elevation on day 3. Reduced glutathione levels were not altered. The observations suggest an important role for alpha-tocopherol in the defense against azide induced free radicals probably including NO and lipid peroxide radicals.

An antiprion effect of the anticytoskeletal drug latrunculin A in yeast.

Prions are infectious aggregation-prone isoforms of the normal proteins, supposedly able to seed aggregation of the normal cellular counterparts. In vitro, prion proteins form amyloid fibers, resembling cytoskeletal structures. Yeast prion [PSI], which is a cytoplasmically inherited aggregated isoform of the translation termination factor Sup35p (eRF3), serves as a useful model for studying mechanisms of prion diseases and other amyloidoses. The previously described interaction between Sup35p and cytoskeletal assembly protein Sla1p points to the possible relationships between prions and cytoskeletal networks. Although the Sup35PSI+ aggregates do not colocalize with actin patches, we have shown that yeast cells are efficiently cured of the [PSI] prion by prolonged incubation with latrunculin A, a drug disrupting the actin cytoskeleton. On the other hand, treatments with sodium azide or cycloheximide, agents blocking yeast protein synthesis and cell proliferation but not disrupting the cytoskeleton, do not cause a significant loss of [PSI]. Moreover, simultaneous treatment with sodium azide or cycloheximide blocks [PSI] curing by latrunculin A, indicating that prion loss in the presence of latrunculin A requires a continuation of protein synthesis during cytoskeleton disruption. The sodium azide treatment also decreases the toxic effect of latrunculin A. Latrunculin A influences neither the levels of total cellular Sup35p nor the levels of chaperone proteins, such as Hsp104 and Hsp70, which were previously shown to affect [PSI]. This makes an indirect effect of latrunculin A on [PSI] via induction of Hsps unlikely. Fluorescence microscopy detects changes in the structure and/or localization of the Sup35PSI+ aggregates in latrunculin A-treated cells. We conclude that the stable maintenance of the [PSI] prion aggregates in the protein-synthesizing yeast cells partly depends on an intact actin cytoskeleton, suggesting that anticytoskeletal treatments could be used to counteract some aggregation-related disorders.

Structure-antiviral activity relationship in the series of pyrimidine and purine N-[2-(2-phosphonomethoxy)ethyl] nucleotide analogues. 1. Derivatives substituted at the carbon atoms of the base.

A series of dialkyl esters of purine and pyrimidine N-[2-(phosphonomethoxy)ethyl] derivatives substituted at position 2, 6, or 8 of the purine base or position 2, 4, or 5 of the pyrimidine base were prepared by alkylation of the appropriate heterocyclic base with 2-chloroethoxymethylphosphonate diester in the presence of sodium hydride, cesium carbonate, or 1,8-diazabicyclo[5,4, 0]undec-7-ene (DBU) in dimethylformamide. Additional derivatives were obtained by the transformations of the bases in the suitably modified intermediates bearing reactive functions at the base moiety. The diesters were converted to the corresponding monoesters by sodium azide treatment, while the free acids were obtained from the diester by successive treatment with bromotrimethylsilane and hydrolysis. None of the PME derivatives in the pyrimidine series, their 6-aza or 3-deaza analogues, exhibited any activity against DNA viruses or retroviruses tested, except for the 5-bromocytosine derivative. Substitution of the adenine ring in PMEA at position 2 by Cl, F, or OH group decreased the activity against all DNA viruses tested. PMEDAP was highly active against HSV-1, HSV-2, and VZV in the concentration range (EC50) of 0.07-2 microg/mL. Also the 2-amino-6-chloropurine derivative was strongly active (EC50 = 0.1-0. 4 microg/mL) against herpes simplex viruses and (EC50 = 0.006-0.3 microg/mL) against CMV and VZV. PMEG was the most active compound of the whole series against DNA viruses (EC50 approximately 0.01-0.02 microg/mL), though it exhibited significant toxicity against the host cells. The base-modified compounds did not show any appreciable activity against DNA viruses except for 7-deazaPMEA (IC50 approximately 7.5 microg/mL) against HIV-1 and MSV. The neutral (diisopropyl, diisooctyl) diesters of PMEA were active against CMV and VZV, while the corresponding monoesters were inactive. The diisopropyl ester of the 2-chloroadenine analogue of PMEA showed substantially (10-100x) higher activity against CMV and VZV than the parent phosphonate. Also, the diisopropyl and diisooctyl ester of PMEDAP inhibited CMV and VZV, but esterification of the phosphonate residue did not improve the activity against either MSV or HIV.

Expression of glycylated tubulin during the differentiation of spermatozoa in mammals.

Using quantitative immunogold analyses of tubulin isoforms we previously demonstrated a unique differential expression of glutamylated tubulin in the flagellum of mouse and man spermatozoa [Fouquet et al., 1997: Tissue Cell 29:573-583]. We have performed similar analyses for glycylated tubulin using two monoclonal antibodies, TAP 952 and AXO 49, directed to mono- and polyglycylated tubulin respectively. Glycylated tubulin was not found in centrioles and cytoplasmic microtubules (manchette) of germ cells. In mouse and man, axonemal tubulin was first monoglycylated and uniformly distributed in all doublets at all levels of the flagellum in elongating spermatids. In human mature spermatozoa axonemal microtubules were enriched in monoglycylated tubulin from the base to the tip of the flagellum. In mouse sperm flagellum a similar gradient of monoglycylated tubulin was also observed in addition to an opposite gradient of polyglycylated tubulin. In both species, monoglycylated tubulin labeling predominated in doublets 3-8 whereas glutamylated tubulin labeling [Fouquet et al., 1997] predominated in doublets 1-5-6. These differential labelings were suppressed after motility inhibition of mouse spermatozoa by sodium azide treatment and in non-motile human spermatozoa lacking dynein arms. The unique distribution of these tubulin isoforms and the known inhibition of motility induced by their specific antibodies are consistent with a complementary role of tubulin glycylation and glutamylation in the regulation of flagellar beating in mammalian spermatozoa.

The Amino-terminal Charge and Core Region Hydrophobicity Interdependently Contribute to the Function of Signal Sequences

We have constructed a series of signal sequence mutants that contain negatively charged amino termini and simplified core regions of varying hydrophobicity levels. This series provides a means of exploring the relative roles of the amino terminus and the hydrophobic core region during transport. The signal peptides with highly hydrophobic core regions support a rapid rate of transport in the presence of a negatively charged amino terminus. We have found that these negatively charged mutants are secreted in a manner similar to the wild-type signal sequence; sodium azide and carbonyl cyanide 3-chlorophenylhydrazone treatments indicate that the negatively charged mutants depend on SecA and the protonmotive force, respectively. These same mutants also demonstrate reduced competition with coexpressed beta-lactamase, reflecting the lower overall affinity for the transport pathway due to the net negative charge at the amino terminus. In addition, the pronounced effects of introducing three negative charges support the conclusion that the two regions function in a concerted manner.

Regional brain effects of sodium azide treatment on cytochrome oxidase activity: a quantitative histochemical study.

The objective of the present study was to determine if regional variation in brain cytochrome oxidase activity was observed following systemic administration of sodium azide. An image analysis system calibrated with internal standards of known cytochrome oxidase activity was used to quantify cytochrome oxidase in histochemically stained brain sections. Rats receiving chronic infusion of sodium azide (400 micrograms/hr), which were sacrificed after two weeks, showed a substantial decrease in brain cytochrome oxidase activity over those infused with saline. All of the 22 regions sampled from telencephalic, diencephalic, and mesencephalic levels, showed a significant activity reduction which ranged between 26% and 37%. The regions that appeared significantly more vulnerable to the sodium azide effects were the mesencephalic reticular formation and the central amygdala, which displayed the largest decrease in activity. In addition, interregional correlations of activity showed a deeply modified pattern of correlative metabolic activity between hippocampal, amygdaloid and cortical areas after azide treatment. The regional effects found were consistent with azide-induced learning and memory dysfunctions.

Chronic Sodium Azide Treatment Decreases Membrane-Bound Protein Kinase C Activity in the Rat Hippocampus

Chronic administration of sodium azide in rats inhibits cytochrome oxidase and produces learning and memory deficits. The present experiment tested the hypothesis that chronic sodium azide treatment might also alter protein kinase C activation. Continuous infusion of sodium azide (400 μg/h, sc) in rats for 2 weeks significantly decreases membrane-bound protein kinase C in hippocampus, but not frontal cortex, temporal cortex, or cerebellum. Since protein kinase C activation is correlated with hippocampus-dependent learning, these results suggest a possible biochemical mechanism for azide-induced impairment of learning.

Mechanism of cell detachment from temperature-modulated, hydrophilic-hydrophobic polymer surfaces

Poly( N-isopropylacrylamide) (PIPAAm), exhibiting a lower critical solution temperature (LCST) at 25 °C in physiological phosphate buffered saline solution (pH 7.4) and at 32 ° C in pure water, was grafted onto the surfaces of commercial polystyrene cell culture dishes. This PIPAAm-grafted surface exhibited hydrophobic surface properties at temperatures over the LCST and hydrophilic surface properties below the LCST. Endothelial cells and hepatocytes attached and proliferated on PIPAAmgrafted surfaces at 37 ° C, above the LCST. The cultured cells were readily detached from these surfaces by lowering the incubation temperature without the usual damage associated with trypsinization. In this case, the optimum temperature for cell detachment was 10 ° C for hepatocytes and 20 ° C for endothelial cells. Cell detachment was partially inhibited by sodium azide treatment, suggesting that cell metabolism directly affects cell detachment. Morphological changes of the adherent cells during cell detachment experiments indicated further involvement of active cellular metabolic processes. Cells detached from hydrophobic-hydrophilic PIPAAm surfaces not only via reduced cell surface interactions caused by the spontaneous hydration of grafted PIPAAm chains, but also by active cell morphological changes which were a function of cell metabolism.

Chronic sodium azide treatment impairs learning of the Morris water maze task

A reduction in the activity of cytochrome oxidase, a respiratory chain enzyme, has been recently identified in mitochondria from blood platelets and postmortem brain tissue from Alzheimer's disease (AD) patients. We have developed an animal model of this deficit in rats by chronic subcutaneous infusion of sodium azide, a selective inhibitor of cytochrome oxidase, delivered via Alzet 2ML4 osmotic minipumps. In previous work, azide-treated rats were impaired in an appetitively motivated spatial learning task, the radial arm maze. In the present investigation, we tested male Sprague-Dawley rats (350-400 g), which were tonically infused with azide or saline, on an aversively motivated spatial task, the Morris water maze. Azide-treated rats were impaired on both acquisition and retention of this task, without showing evidence of a motor impairment. Thus, the present results are consistent with previous findings showing that chronic azide treatment produces a learning and memory deficit. These findings strengthen the hypothesis that azide treatment in rats produces a useful animal model of some aspects of AD.

Chlorination of cellulose with sulfuryl chloride in lithium chloride-dimethylacetamide and azidation of the products.

ABSTRACT: Products containing chlorine and chlorosulfate groups were prepared by the chlorination of cellulose powder with sulfuryl chloride in a mixture of lithium chloride and N, N-dimethylacetamide in the presence of pyridine. The sodium iodide treatment of this product yielded chlorodeoxycellulose with a maximum d. s. of 1.8 by chlorine, which was introduced at C-6 and C-3. Chlorosulfate groups were rather stable to acidic or neutral water, but unstable in alkaline water and were converted into hydroxyl groups or cyclic sulfates. They were replaced by azide and chloride ions or converted into cyclic sulfates by the sodium azide treatment in N, N-dimethylformamide at temperatures of 20 to 70°C. The same treatment of the chlorination product at 90°C resulted in the replacement of chlorine by azide and that at 110°C gave a product with a high d. s. of 1.1 by azide.

Effect of Mutagenic Treatments on Somaclonal Variation in Wheat (Triticum aestivum L.)

AbstractDifferent wheat genotypes were treated with gamma‐rays, sodium azide (SA) and EMS before tissue culture and immature embryos from M1 plants or plants shortly after exposure to gamma‐rays were used to initiate callus culture. Thousands of plants were regenerated and used to investigate the effect of mutagenic treatments on the regenerated plants and somaclonal variation in the M3R2 and M2R2 generations. The results showed that mutagen‐induced damage in terms of reduction in plant height, fertility and spike length were not outstanding in the regenerated plants as compared with the untreated control. In the M3R2 generation, only SA treatment had significantly higher frequencies of somaclonal variations than the control. Increases in the variation frequencies were observed when explant embryos were irradiated with 2.5 and 5 gy gamma‐rays and the highest frequency appeared when embryos were exposed to 5 gy gamma‐rays on the 5th day after anthesis. Increased variation spectra also resulted from mutagenic treatments and most of the variants recovered were unsuitable for plant improvement.

Mutagenic activity of 3-azido-1,2-propanediol and sodium azide applied to sugar cane callus cells

Chlorophyll deficient plantlets were obtained after the treatment of sugar cane callus cells with 3-azido-l,2-propanediol (AG) and sodium azide with approximately the same frequency. Both mutagenic agents postponed the regeneration of plantlets from calli, the total number of regenerated plantlets following the AG treatment being, however, higher than after the sodium azide treatment and control.

Biological and genetic effects of combined treatments of sodium azide, gamma rays and EMS in barley

Dry seeds of diploid barley were subjected to mutagenic treatments of sodium azide, gamma rays and EMS alone or in combination. Damage (reduction in seedling height, plant height and fertility), the frequency of chimeras in the M 1 generation, and the frequency of chlorophyll-deficient mutations as well as morphological mutations in the M 2 generation induced by combined treatments were greater than those by either of the single treatments. Synergistic increase in the frequency of chimeras, chlorphyll-deficient mutations and morphological mutations were observed in both sodium azide post-irradiation treatments and pre-EMS treatments; interaction among the mutagens in the treatment combinations on M 1 damage was generally subtractive. An 8- to 16-hr soaking period of irradiated seeds in distilled water prior to sodium azide treatment significantly increased chlorophyll mutation frequency, as compared to that from the non-soaking treatment. Damage and frequency of chimeras, chlorophyll mutations and morphological mutations were consistently reduced by the soaking treatment in sodium azide plus EMS treatments.

Synthesis of a Chiral Hexane-1,6-Dinitrile from D-Glucose

Abstract Using well-established methodology, D-glucose was transformed into a tribenzoyloxy cyclohexanone oxime derivative. Sodium azide treatment permitted the regiospecific exchange of the benzoyloxy group at a α-position relative to ketone oxime. Under standard Beckmann conditions the α-azido was oxime cleaved to provide an ω-dicyano compound.