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The thermolysis of 2-azido-2-tert-butyl-2H-selenochromenes (2a, b), which were easily obtained by the reaction of the corresponding 1-benzoselenopyrylium salts (1) with sodium azide, resulted in a ring expansion to give the novel stable 1,3-benzoselenazepines (5a, b) with denitrogenation in good yields.
Earlier investigations from our laboratory demonstrated that human term placental peroxidase (HTPP) is capable of metabolism of xenobiotics and endogenous compounds. In this study, purified HTPP was found to bioactivate 2-aminofluorene (2-AF) in the presence of H2O2. 2-AF oxidation was studied spectrophotometrically while radiometry was employed to assess the bioactivation. The rate of oxidation and covalent binding to protein and DNA was dependent upon the pH of the reaction medium and the concentration of 2-AF, the enzyme, and H2O2. To observe maximal enzyme velocity of oxidation, the presence of 16.5 microM H2O2, 100 microM 2-AF, 37 micrograms of the enzyme protein/ml, and pH 7.2 was required. Under optimal assay conditions, the range of specific activity between 130 and 165 nmol of 2-AF oxidized/min/mg HTPP was observed. Using similar assay conditions, the magnitude of covalent binding of [3H]-2-AF to protein (BSA) and calf thymus DNA was found to be about 508 pmol bound/min/mg HTPP/mg BSA and 84 pmol bound/min/mg HTPP/mg DNA, respectively. Potassium cyanide and sodium azide, the known inhibitors of different peroxidases, significantly blocked both the oxidation and covalent binding of 2-AF in a dose dependent manner. These results strongly suggest that peroxidase may be one of the important pathways responsible for the bioactivation of arylamines in human term placenta.
To test the activity of P-170 glycoprotein in isolated hepatocytes, a method has been developed employing the fluorescent dye rhodamine 123 (R-123). The uptake of R-123 by both freshly isolated and 4-hr-plated hepatocytes depends on dye concentration, time of incubation, and cell number. The efflux of R-123 from cells is inhibited by sodium azide and by verapamil. In standard conditions the efficiency of efflux of R-123 from cells correlates with the relative amount of immunoblottable glycoprotein. The method has been applied to detection of P-170 activity in hepatocytes from animals of different ages as well as from carcinogen-treated animals. The proposed assay appears a simple and adequate tool for the functional assessment of multidrug transporter in liver.
The effect in vitro of some cytoplasmic structure and function inhibitors on the different stages of rabies virus infection was investigated. Treatment of fibroblasts (CER) and human neuroblastoma cells (IMR-32) with substances acting on low pH intracellular compartments (methylamine and monensin) prevented rabies virus genome delivery in the cytosol. An early inhibition of viral infection was also obtained in the presence of B and D cytochalasins and trifluoperazine which interact with microfilament structures. Treatment with colchicine and vinblastine did not affect rabies multiplication, suggesting that microtubules are not involved in this process. However, the multiplication of prebound virions did not take place in the presence of inhibitors of oxidative phosphorylation (sodium azide and CCCP) and of glycolysis (2-deoxy-D-glucose) indicating that rabies virus replication is largely energy-dependent in both host cells examined.
Cellular interactions control lymphocyte localization within salivary gland tissues and contribute to the immune defense of oral surfaces. We examined lymphocyte adherence to cultured parotid cells using an in vitro assay and found good correlation with previously reported binding to parotid gland frozen sections. Thoracic duct lymphocytes (TDL) bound to parotid cells in greater numbers than thymocytes (74 vs 11 cells/mm2). B cells showed preferential adherence compared to T cells (75% vs 28%). TDL binding was inhibited by sodium azide or cytochalasin B (60% and 80%, respectively). EDTA inhibition (63%) was restored by replacing calcium (9%) but not magnesium (65%). Binding was inhibited by fucoidin or phosphomannan (approximately 70%). Fibronectin peptides had no effect. Culture supernatants were inhibitory for TDL adherence (60%), suggesting that molecules involved in lymphocyte localization may be shed and that parotid cell cultures will be useful for ligand isolation and characterization.
Maintenance of cell calcium homeostasis and transepithelial transport of this cation require its extrusion from the cell against a steep electrochemical gradient. Because it has been proposed that a membrane Ca-ATPase activated by micromolar concentrations of Ca2+ prevailing in the cell participates in these processes, we attempted in this study to determine whether such an enzyme is present in the rabbit nephron. A magnesium-dependent ATPase, maximally activated by Ca2+ (Ca-Mg-ATPase) concentrations between 1.1 and 2.3 microM (apparent Km = 0.3-0.4 microM), was found in all segments of the nephron. Ca-Mg-ATPase (pmol.mm-1.h-1) was highest in the distal convoluted tubule (243) and cortical collecting tubule (208), intermediate in the proximal convoluted tubule (140) and medullary thick ascending limb of Henle's loop (135), and lower in the pars recta (97), cortical thick ascending limb (50), and medullary collecting tubule (51). The enzyme was insensitive to ouabain and vanadate, but was inhibited by ruthenium red in a dose-dependent manner (Ki congruent to 2.10(-6) M). Sodium azide, an inhibitor of mitochondrial ATPase, did not affect Ca-Mg-ATPase, suggesting that the enzyme was located in the plasma membrane. The Ca-Mg-ATPase activity measured in most segments of the rabbit nephron in this study appears sufficient to account in theory for the active component of the unidirectional (lumen-to-bath) calcium flux found in the corresponding region of the nephron with in vitro single tubule microperfusion techniques.
Solanum melongena fruit juice contains peroxidase activity of the order of 0.125 IU/mL. A method for the 11-fold purification of the enzyme was developed. The Km values of the peroxidase for the substrates guaiacol and hydrogen peroxide were 6.5 mM and 0.33 mM, respectively. The pH and temperature optima were 5.5 and 84 degrees C, respectively using guaiacol as the substrate. Sodium azide and phenyl hydrazine inhibited the enzyme competitively.
Subcellular localization of meta-tetra (hydroxyphenyl) chlorin (mTHPC) in HT29 human colon adenocarcinoma cells has been studied by means of fluorescence microscopy. The observed diffuse intracellular distribution of mTHPC fluorescence outside the nucleus indicates general staining of cellular organelles. No changes in dye fluorescence pattern are evident during and immediately after cell illumination. Alternatively, the changes in mTHPC fluorescence pattern are observed upon subsequent cell incubation, and are characterized by the appearance of distinct bright fluorescence zones. Reaching a maximum 1 h after illumination, modifications of the fluorescence pattern then gradually disappear in parallel with the formation of plasma membrane blebs, suggesting that cell necrotic lysis is taking place. Photosensitized damage to mitochondria and the Golgi apparatus has been studied using fluorescent probes 1 h after irradiation, the stage of extensive cytoplasm vacuolization, and reveal alterations of these organelles. Changes in the mTHPC fluorescence pattern and mTHPC photocytotoxicity, as measured by the MTT test 24 h after illumination, are inhibited by sodium azide, a singlet oxygen quencher.
Biomolecular motors such as F1-adenosine triphosphate synthase (F1-ATPase) and myosin are similar in size, and they generate forces compatible with currently producible nanoengineered structures. We have engineered individual biomolecular motors and nanoscale inorganic systems, and we describe their integration in a hybrid nanomechanical device powered by a biomolecular motor. The device consisted of three components: an engineered substrate, an F1-ATPase biomolecular motor, and fabricated nanopropellers. Rotation of the nanopropeller was initiated with 2 mM adenosine triphosphate and inhibited by sodium azide.
In a previous report chromate potentiated the mutagenicity of sodium azide, apparently by affecting repair and/or replication of DNA. Further evidence in support of such a mechanism for chromate potentiation is reported here. Chromate does not react directly with azide or its major mutagenic metabolite, azidoalanine, eliminating such reactions as possible mechanisms for potentiation. Further, azide was unable to potentiate the mutagenicity of chromate in Salmonella typhimurium strain TA104, which is sensitive to chromate mutagenicity but not to azide. Thus, it appears that the potentiation is not due to an action of azide in modulating chromate mutagenicity. Finally, the interaction was not altered by deficiency in recA gene product in S typhimurium GW19, nor by enhancement of SOS repair in the pKM101 containing strain TA100. Thus, induction of recA-dependent functions seems to play no role in the comutagenic actions of chromate. The simplest explanation for potentiation seems to be that chromate is able either to limit error-free recovery from azide-induced DNA damage or to promote error-prone repair or error-prone processing at sites of lesions.
Experiments to elucidate the mechanism by which Pneumocystis carinii transports glutamine, leucine, and serine were performed in this study. Uptake of all three radiolabeled amino acids exhibited first-order, saturation kinetics as extracellular substrate concentrations were increased, thus ruling out simple diffusion and indicating carrier-mediated transport. Kinetic analyses of amino acid uptake and the results of competitive inhibition experiments suggested that leucine, serine, and glutamine were taken up via a common transporter system. The uptake of serine was examined in greater detail to characterize the nature of the carrier. Serine uptake was not affected by N, N'-dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl hydrazone, ouabain, gramicidin, valinomycin, sodium azide, salicylhydroxamine acid (SHAM), iodoacetate, iodoacetate plus SHAM, KCN, and azide. Thus serine uptake did not require sodium or energy from ATP, an electrochemical proton gradient or a membrane potential across the cell surface (i.e., proton-motive force). Serine uptake was dependent on glucose in the extracellular compartment. In the presence of glucose, serine uptake was inhibited by chloramphenicol but not cycloheximide. The results from these experiments are most consistent with facilitated diffusion as the mechanism. After 30 min of incubation, most of the radioactivity was in the cellular soluble fraction. In most cases, incorporation into the extractable total lipids and the remaining particulate cellular components were detectable after this incubation period.
An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM L-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN3), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN3 as a blocker of the respiratory chain was indispensible, because without NaN3 most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities.
In this work we describe the ability of living Crithidia deanei to hydrolyze extracellular ATP. In intact cells at pH 7.2, a low level of ATP hydrolysis was observed in the absence of any divalent metal (0.41+/-0.13 nmol P(i) h(-1) 10(7) cells(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg(2+)-dependent ecto-ATPase activity was 4.05+/-0.17 nmol P(i) h(-1) 10(7) cells(-1). Mg(2+)-dependent ecto-ATPase activity increased linearly with cell density and with time for at least 60 min. The addition of MgCl(2) to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.93+/-0.26 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.26+/-0.03 mM. ATP was the best substrate for this enzyme; other nucleotides, such as ITP, GTP, UTP and CTP, produced lower reaction rates. In the pH range from 6.6 to 8.4, in which the cells were viable, the acid phosphatase activity also present in this cell decreased, while the Mg(2+)-dependent ATPase activity did not change. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, vanadate, molybdate, sodium fluoride and tartrate. To confirm that this Mg(2+)-dependent ATPase was an ecto-ATPase, we used the impermeant inhibitor 4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The cell surface location of the ATP-hydrolyzing site was also confirmed by cytochemical analysis.
In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53+/-0.12 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 5.24+/-0.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2), SrCl(2), and ZnCl(2). The apparent K(m) for Mg-ATP2- was 0.53+/-0.11 mM. The optimum pH for the T. rangeli Mg-dependent ecto-ATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph.
Potassium tellurite media gave the highest detrimental effects on the generation number of all tested faecal streptococci species. This applies to all investigated vehicles. However, drastic tellurite effects became more conspicuous with certain vehicles than with others. This is particularly true with acid and alkaline foods and water, especially with strains like Streptococcus bovis, Str. faecium, and Str. durans. Again, tellurite becomes more influencial with regard to old cells rather than young cells in the logarithmic phase. On the other hand, the use of thallous acetate as a concentration agent secured better results than either the tellurite or azide almost with all tested vehicles. Yet, sodium azide may be regarded as holding an intermediate position between tellurite and thallous acetate in terms of vehicles, Streptococcus species, and age of pollution. A lower number of generation resulted with all tested faecal streptococci species, using liquid media bearing more than one concentrating agent. This applied to water and milk examination.
We describe a fatality resulting from the ingestion of sodium azide. Initial routine toxicological screening of the blood showed a negative result, but pathological findings as well as findings at the scene itself led to the search for azide. The azide was quantitated in all available postmortem samples (blood, stomach contents, liver, kidney, and bile) and was present in all of these matrices in the following concentrations (micrograms per milliliter or micrograms per gram): 262, 754, 14, 205, and 1283, respectively. The method is based on precolumn derivatization with 3,5-dinitrobenzoyl chloride followed by isocratic high-performance liquid chromatography and photodiode array detection. Methemoglobin and cyanide content in the blood were also elevated.
A soluble, sodium-nitroprusside-stimulated guanylate cyclase as been purified from bovine lung by DEAE-cellulose chromatography, ammonium sulfate precipitation, chromatography on Blue Sepharose CL-6B and preparative gel electrophoresis. Apparent homogeneity was obtained after at least 7000-fold purification with a yield of 3%. A single stained band (Mr 72000) was observed after gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme migrated as one band also under non-denaturing conditions in acrylamide gels (5-12%). The mobility of this band corresponded to an Mr of 145000. The enzyme sedimented on sucrose gradients with an S20, w of 7.0 S. Gel filtration yielded a Stokes' radius of 4.6 nm. These data suggest that the enzyme has an Mr of approximately 150000 and consists of two, presumably identical, subunits of Mr 72000. Sodium nitroprusside stimulated the purified enzyme 15-fold and 140-fold to specific activities of 8.5 and 15.7 mumol of cGMP formed min-1 mg-1 in the presence of Mn2+ and Mg2+, respectively. Formation of cGMP was proportional to the incubation time and to the amount of enzyme added. The stimulatory effect of sodium nitroprusside was half-maximal at about 2 microM, was observed immediately after addition and could be reversed either by dilution or by removal of sodium nitroprusside on a Sephadex G-25 column. The purified enzyme in the absence of catalase was stimulated by sodium nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine and 3-morpholino-sydnonimine and in the presence of catalase by sodium nitrite and sodium azide. In the presence of Mn2+ and sodium nitroprusside, the purified enzyme catalyzed the formation of cAMP from ATP at a rate of 0.6 mumol min-1 mg-1.
A profound decrease in activity of the mitochondrial enzyme cytochrome oxidase in blood platelets is a recently identified concomitant of Alzheimer's disease (AD). We investigated a possible pathogenic link between this finding and the symptoms of AD by mimicking this mitochondrial enzyme deficiency in rats. Rats were infused chronically with a selective inhibitor of cytochrome oxidase, sodium azide, or with saline delivered via subcutaneously implanted osmotic minipumps. The azide treatment impaired both spatial and nonspatial learning. Further, the azide treatment inhibited a low-threshold form of hippocampal long-term potentiation, primed burst potentiation. The behavioral deficits were not secondary to a sensory or motor impairment. Thus, chronic azide treatment of rats models some characteristics of AD.