Azide Treatment Research Articles

Human serum catalase decreases endothelial cell injury from hydrogen peroxide

Serum from normal human subjects contained variable amounts of catalase activity, which was inhibitable by heat, azide, trichloroacetic acid (TCA), or aminotriazole treatment. Serum also decreased hydrogen peroxide (H2O2) concentrations in vitro and H2O2-mediated injury to cultured endothelial cells. By comparison, heat-, azide-, TCA-, or aminotriazole-treated serum neither decreased H2O2 concentrations in vitro nor reduced H2O2-mediated damage to endothelial cells. We conclude that serum catalase activity can alter H2O2-dependent reactions. We speculate that variations in serum catalase activity may alter individual susceptibility to oxidant-mediated vascular disease or be a factor when added to test systems in vitro.

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.

Induction of apoptosis (programmed cell death) in human leukemic HL-60 cells by inhibition of RNA or protein synthesis.

Apoptosis is regarded as a suicidal cell response since the dying cell appears to be an active participant. Previous studies have shown that apoptosis of various murine cell types, induced by a variety of stimuli, required RNA and/or protein synthesis. However, when human promyelocytic leukemia HL-60 cells were induced to undergo apoptosis by treatment with the calcium ionophore A23187 or microtubule-disrupting agents, in the presence of inhibitors of macromolecular synthesis, apoptosis of these cells was neither abrogated nor delayed. Furthermore, the presence of either cycloheximide, an inhibitor of protein synthesis, or actinomycin D, an RNA synthesis inhibitor, alone was found to induce large scale apoptosis of these cells. Apoptosis in these cells was characterized by cell and chromatin condensation followed by nuclear and DNA fragmentation. In common with many other studies, this DNA fragmentation was found to have an approximately 200-bp multiple pattern, which is consistent with the activation of an endogenous endonuclease which cleaves at internucleosomal sites. Calcium-dependent endonuclease activity of this type was also detected in the isolated nuclei of untreated HL-60 cells. The morphologic and biochemical changes characteristic of apoptosis were found to precede cell death, as measured by trypan blue uptake and were completely distinct from death caused by toxic stimuli such as azide, ethanol, or heat treatment. Similar experiments with six other human cell lines confirmed that this phenomenon was not peculiar to the HL-60 cell line. These results suggest that certain dividing cell populations do not require RNA or protein synthesis to undergo apoptosis and further, that continuous transcription and translation of some regulatory protein(s) may be required to maintain control over the apoptotic "machinery" of such cells.

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.

Differential metabolism of sodium azide in maize callus and germinating embryos

Sodium azide is a potent mutagen of maize ( Zea mays L.) kernels that may have potential as a point mutagen for inducing biochemical mutations in maize tissue cultures. Azide mutagenicity was evaluated in friable, embyogenic maize callus and a nonregenerable maize suspension culture by determining the number of resistant variant cell lines able to grow on media containing inhibitory concentrations of lysine plus threonine (LT). The number o0f LT-resistant variants selected from either culture type did not increase in response to azide treatment. In addition, there was no increase in somatic mutations in more than 100 plants regenerated from azide treated LT-resistant lines. The levels of mutagenic metabolite of azide (presumably azidoalanine), were determined by bioassay in the two azide-treated maize callus types and compared to levels of mutagenic metabolite in embryos isolated from azide-treated kernels. The two types of maize tissue cultures and isolated embryos contained similar levels of mutagenic metabolite 4 h after azide treatment indicating similar uptake and conversion of azide to mutagenic metabolite in the three tissues. Mutagenic metabolite in azide-treated embryos did not significantly decrease after 40 h. However, mutagenic metabolite levels in both azide-treated tissue cultures decreased to near background levels within 20 h providing evidence for rapid metabolism of the azide mutagenic metabolite. The lack of evidence for azide mutagenicity in maize callus and its known potent mutagenicity in kernels appears to be associated with specific differences in azide metabolism between callus tissues and kernel embryos.

Synthesis ofN-Trifluoroacetyl Derivatives of Enantiomeric 2,5,6-Trideoxy-5-Amino Hexoses from Non-Carbohydrate Precursors

Abstract The D-lyxo and D-ribo methyl-N-trifluoroacetyl-2,5,6-trideoxy-5-amino hexofuranosides (13) and (14) are accessible from carbohydrate-like acyclic C7 products 3 and 5, whereas the 3-C-methyl analog of 13 is prepared in similar way from 15. Azide treatment of the toluensul-phonate ester at position 4 of methyl-2,6-dideoxy-3-C-methyl-L-ribo hexopyranoside (21), using hexamethylphosphoric acid triamide as cosolvent, gives rise to a mixture of azido derivatives in which prevails the arabino material (24), formed from 21 via inversion of configuration at positions 4 and 5.Product 24 gives rise to the 5-amino sugar derivative 28. The enantiomers of 13 and 14 are accessible from D-allo threonine and L-threonine, respectively.

Differential arrest of secretory protein transport in cultured rat hepatocytes by azide treatment.

The effect of reduced cellular ATP content on intracellular transport of two secretory proteins, albumin and haptoglobin, in isolated rat hepatocytes was studied. The cells were labeled with [35S]methionine and the cellular ATP content was then rapidly reduced to different stable levels by incubation with azide at different concentrations (2.0-10 mM). The amount of the radioactively labeled secretory proteins in the cells and in the medium after 150 min of incubation was determined by immunoprecipitation followed by gel electrophoresis, fluorography, and densitometry. At progressively lower ATP levels, down to 50% of normal, the protein secretion was unaffected, whereas at even lower levels an increasing portion of the proteins remained in the cells; at 30 and 10% of normal ATP level, 25 and 75% of albumin, respectively, was arrested intracellularly. Analysis of the carbohydrate structure of intracellularly arrested haptoglobin showed that in cells with an ATP level of approximately 30% of normal, the majority of haptoglobin molecules (55%) were fully or partially resistant to endoglycosidase H. This result indicates that exit from the medial and/or the trans part of the Golgi complex (GC) was inhibited under these conditions. It also shows that the protein had accumulated in the GC, since under normal conditions the fraction of the intracellular haptoglobin that is endoglycosidase H resistant is approximately 10%. By similar criteria it was found that at ATP levels below 10% of normal transport of haptoglobin from the endoplasmic reticulum to the medial GC (and possibly also to the cis GC) as well as from the trans GC to the medium were blocked.

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.

Relative effectiveness and efficiency of single and combination treatments using gamma rays and sodium azide in inducing chlorophyll mutations in rice.

Seeds of rice cultivars Jaya, IET 5656 and Fujiminori were presoaked for 24 hours and then (a) irradiated with 4, 6, 8, 10 and 12 KR gamma rays, (b) treated with 0.001, 0.002, 0.004 and 0.005 molar solutions of sodium azide at pH 3 for 4 hours, or (c) subjected to combinations of the above with the view to study their relative effectiveness and efficiency in inducing chlorophyll mutations in rice. In all three varieties azide treated entries showed a higher frequency of chlorophyll mutants than did the gamma ray treated entries, under both single and combination treatments. In the variety Jaya sodium azide treatments showed a higher efficiency and effectiveness than gamma rays and combination treatments and they were more or less similar in both single and combination treatments in the other two varieties. The indica cultivars Jaya and IET 5656 were more sensitive than the japonica cultivar, Fujiminori to producing chlorophyll mutations. Sodium azide appears to be more efficient than gamma rays alone or in combination.

Initial adhesion of murine fibroblasts to collagen and fibronectin occurs by two mechanisms.

The adhesion of Balb/c 3T12 cells to fibronectin (FN) and to denatured (DC) or native (NC) collagen is differentially sensitive to divalent cations and to sodium azide. Short-time adhesion (10 min) to FN requires either Mg2+ or Mn2+, whereas only Mn2+ stimulates attachment to DC and NC. Azide treatment only slightly affects adhesion of cells to FN, but strongly inhibits cell attachment to DC and NC. Attachment to any of these substrata is unaffected by monensin and by treatment of the cells with an intracellular fraction, making unlikely the possibility that molecules released by secretion or cell lysis participate in the adhesive process. Soluble collagen inhibits the adhesion of cells to DC and NC, but does not affect adhesion to FN. Finally, rabbit antiserum against collagen binding proteins inhibits cell attachment to NC and DC; the cells, however, attach normally to FN in presence of this antiserum. Taken together, our results support the view that 3T12 cells attach directly to native or denatured collagens and that FN is not required for this process.

Alteration in glyceollin synthesis and antigenic patterns after chemical induction of resistance in soybean to Macrophomina phaseolina

Sodium azide was found to be most effective of the six metabolic inhibitors tested in reducing charcoal rot disease of soybean (cv. Soymax) caused by Macrophomina phaseolina. Glyceollin production also increased significantly after induction of resistance by sodium azide treatment. Cross-reactive antigens were detected in purified preparations from mycelia of M. phaseolina with antisera of soybean roots by immunodiffusion and immunoelectrophoretic tests. An antigenic dispartity was noticed in the susceptible cultivar (cv. Soymax) after chemical induction of resistance. The changes in antigenic pattern and their involvement in induced resistance of soybean to M. phaseolina are discussed.

Studies of phagocytosis in chronic granulomatous disease.

Abnormal phagocyte function in chronic granulomatous disease (CGD) is associated with decreased bactericidal activity. Ingestion of serum-opsonized organisms is reported to be normal in these patients. We previously showed that in CGD the expression of C3b receptors (CR1) on polymorphonuclear leukocytes (PMNs) is significantly depressed. In this study, we compared the phagocytic activity of the PMNs from normal healthy controls with that of CGD patients and one individual with myeloperoxidase (MPO) deficiency. The ingestion of sheep erythrocytes (E) by PMNs adherent to a glass surface was examined; the E were coated either with excess IgG (E-IgG) or with C3b plus limited IgG (EAC3b-IgG). The PMNs, both in CGD and in MPO deficiency, ingested E-IgG and EAC3b-IgG at levels markedly above normal. C3b-coated erythrocytes were not phagocytosed. Preincubating the PMNs with sodium azide, which blocks MPO, or catalase, a scavenger of H2O2, caused a marked increase in phagocytosis by normal PMNs. Azide had a variable effect on PMN activity in CGD and no effect on the activity in the subject with MPO deficiency. Even in the presence of azide, the ingestion of EAC3b-IgG by the PMNs from the CGD patients was significantly greater than that seen in paired normals [mean phagocytic index (PI), 2.13 for CGD vs. 1.48 for normals; P less than 0.05 by the paired sample t test]. Similar results were obtained with ingestion of E-IgG. Notably, ingestion of serum-opsonized Candida organisms (relatively nondegradable particles) was markedly above normal with CGD PMNs and, in normal PMNs, azide treatment also evoked an increase. In addition, rosette formation of the adhered PMNs with E-IgG was enhanced with CGD and the azide-treated normal PMNs. We demonstrated that this increased activity was not the result of increased Fc receptor (FcR) number, as determined from the binding of a monoclonal anti-FcR antibody. Both the E-IgG rosette formation and the ingestion by CGD PMNs were abrogated in the presence of an H2O2-generating system. In contrast, the phagocytic activity of MPO-deficient PMNs was not altered by exogenous H2O2. These findings suggest that cellular products generated by the H2O2-MPO-halide system down-regulate the rosette-forming and phagocytic activity of PMNs from normal healthy individuals, but not that from CGD and MPO-deficient patients.

Metabolic inhibitors block anaphase A in vivo.

Anaphase in dividing guard mother cells of Allium cepa and stamen hair cells of Tradescantia virginiana consists almost entirely of chromosome-to-pole motion, or anaphase A. Little or no separation of the poles (anaphase B) occurs. Anaphase is reversibly blocked at any point by azide or dinitrophenol, with chromosome motion ceasing 1-10 min after application of the drugs. Motion can be stopped and restarted several times in the same cell. Prometaphase, metaphase, and cytoplasmic streaming are also arrested. Carbonyl cyanide m-chlorophenyl hydrazone also stops anaphase, but its effects are not reversible. Whereas the spindle collapses in the presence of colchicine, the chromosomes seem to "freeze" in place when cells are exposed to respiratory inhibitors. Electron microscope examination of dividing guard mother cells fixed during azide and dinitrophenol treatment reveals that spindle microtubules are still present. Our results show that chromosome-to-pole motion in these cells is sensitive to proton ionophores and electron transport inhibitors. They therefore disagree with recent reports that anaphase A does not require a continuous supply of energy. It is possible, however, that anaphase does not directly use ATP but instead depends on the energy of chemical and/or electrical gradients generated by cellular membranes.

A simplified method for determination of tritiated thymidine incorporation into cells from tissue in soft agar culture.

We designed a simple method for in vitro chemosensitivity testing of tritiated thymidine incorporation by cells from tumor tissue plated in soft agar. Cells from tumor tissue were cultured in a plastic dish, of which a Millipore membrane had been sealed to the underside, containing two layers of agar. After labeling the cells with tritiated thymidine, the dishes were placed in a boiling water bath to solubilize the agar. The labeled cells could be readily collected on the membrane by evacuating the solubilized medium through membrane filters sealed underside of the dish. Using this system, 11 (55 per cent) of 20 tumor specimens from 20 patients with various carcinomas showed an incorporation of over 200 dpm above background and 80 per cent or greater inhibition of thymidine uptake by sodium azide treatment. This method offered several advantages over the thymidine incorporation assay, including technical simplicity and a shorter time course.

Induction of proteinase K-sensitive sites and M. luteus endonuclease-sensitive sites in DNA of barley embryos by sodium azide

DNA damage induced in germinating barley embryos by mutagenic and sublethal doses (0.1–2 mM, 2 h) of sodium azide, applied at pH 3, was measured by alkaline elution. Isolated nuclei were lysed at a high pH with either 2% SDS or 2 M NaCl on polyvinyl chloride filters and digested with proteinase K or with Micrococcus luteus endonuclease prior to elution. The azide treatments resulted in a dose-dependent increase of proteinase K-sensitive sites and an appearance of Micrococcus luteus endonuclease-sensitive sites. These sites were detected as DNA single-strand breaks after digestion of the DNA with either one or both of the enzymes. The two types of lesion were additive and occurred in a ratio of about 1:1. The additive effect suggested independent origin for the two types of lesion. Breaks independent of proteinase K digestion appeared only when DNA was analysed 24 h after the action of azide. The nature and significance of these DNA lesions are discussed.

A Mg 2+-independent high-affinity Ca 2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg 2+

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca 2+-stimulated ATPase activity in the absence of Mg 2+. The Ca 2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity ( K m = 0.4 μM ) and a low affinity ( K m = 0.6 mM ) for Ca 2+. Distribution of these high-affinity and low-affinity Ca 2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg 2+ also stimulates the ATPase in the absence of Ca 2+. Unlike the Mg 2+-ATPase and low-affinity Ca 2+-ATPase, the plasmalemmal high-affinity Ca 2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca 2+-ATPase is noncompetitively inhibited by Mg 2+ with respect to Ca 2+ stimulation. Such an inhibitory effect of Mg 2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca 2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg 2+-independent, high-affinity Ca 2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg 2+-dependent, high-affinity Ca 2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca 2+-pump.

Influence of temperature during and after sodium azide treatment on M 1 damage and M 2 chlorophyll mutation in barley ( Hordeum vulgare L.)

After sodium azide (NaN 3) treatment at different temperatures (5–35°C), barley seeds were exposed to temperature at 5–35°C for 24 hr. With proportionate increase in NaN 3-treatment temperature, delay of germination and yield of chlorophyll mutations increased but mutation frequency and the delay of germination were reduced by post-exposure to higher temperatures for each NaN 3 treatment: temperature group. The germination delay was virtually eliminated when the post-treatment was carried out at pH 7. The yield of chlorophyll mutations ranged from 0.61 to 6.86% for M 2 seedlings (a 10-fold difference), and from 7.9 to 28.9% for M 1 spikes. The results indicate an interaction between the mutagenic action of NaN 3 and repair of the induced lesions during germination.

Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death.

Dexamethasone and corticosterone kill mouse thymocytes, as measured by eosin uptake, after several hours of in vitro incubation. This killing requires RNA and protein synthesis, because it is inhibited by cycloheximide, emetine, or actinomycin D. An earlier event than cell death is the extensive fragmentation of nuclear DNA into oligonucleosomal subunits; this fragmentation also requires RNA and protein synthesis. The DNA cleavage results from the action of an endonuclease that preferentially cleaves DNA in the linker regions between nucleosomes. This endonuclease is found constitutively in the nuclei of thymocytes and some other cells, and requires calcium and magnesium ions for its activation; if isolated fresh thymocyte nuclei are incubated with these ions, as much as 77% of their DNA is cleaved within 90 min. Thus, the protein for which synthesis is necessary for glucocorticoid-induced thymocyte death is not the endonuclease itself, but is in some way involved in its activation; we suggest that it may be part of a system for transporting calcium into the nucleus. The endonuclease is inhibited by zinc, which also prevents thymocyte death. It appears that glucocorticoids cause thymocyte death by activating an enzyme that rapidly and extensively degrades DNA. This "death from within" is biochemically and morphologically different from toxic or accidental cell death, such as that induced by azide, heat, or antibody and complement treatment. Although mature T cells also contain the endogenous endonuclease, they lack the glucocorticoid-inducible mechanism for activating it, and are thus glucocorticoid-resistant.

Germination of Setaria chevalieri caryopses

SummaryGermination studies were made on Setaria chevalieri caryopses (seeds). The seeds imbibed readily upon moist incubation. An after‐ripening period which followed a cyclic patlern was necessary for maximum germination. Freshly harvested seed germinated in the presence of light, but only very sporadically in the dark. The germination of dark incubated seed was improved if the seeds were subsequently exposed to light. This photodormancy became less pronounced with dry storage. Treatment with red light increased germination. but was reversed by far‐red light suggesting that a phytochrome system operates in the seeds. Sodium azide treatments did not stimulate germination in the dark but were effective in the presence of light.