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PubMed1985

Novel ATP-dependent calcium transport component from rat liver plasma membranes. The transporter and the previously reported (Ca2+-Mg2+)-ATPase are different proteins.

An ATP-dependent calcium transport component from rat liver plasma membranes was solubilized by cholate and reconstituted into egg lecithin vesicles by a cholate dialysis procedure. The uptake of Ca2+ into the reconstituted vesicles was ATP-dependent and the trapped Ca2+ could be released by A23187. Nucleotides, including ADP, UTP, GTP, CTP, GDP, AMP, and adenyl-5'-yl beta, gamma-imidophosphate, and p-nitrophenylphosphate did not substitute for ATP. The concentration of ATP required for half-maximal stimulation of Ca2+ uptake into the reconstituted vesicles was 6.2 microM. Magnesium was required for calcium uptake. Inhibitors of mitochondrial calcium-sequestering activities, i.e. oligomycin, sodium azide, ruthenium red, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and valinomycin did not affect the uptake of Ca2+ into the vesicles. In addition, strophanthidin and p-chloromercuribenzoate did not affect the transport. Calcium transport, however, was inhibited by vanadate in a concentration-dependent fashion with a K0.5 of 10 microM. A calcium-stimulated, vanadate-inhibitable phosphoprotein was demonstrated in the reconstituted vesicles with an apparent molecular weight of 118,000 +/- 1,300. These properties of Ca2+ transport by vesicles reconstituted from liver plasma membranes suggest that this ATP-dependent Ca2+ transport component is different from the high affinity (Ca2+-Mg2+)-ATPase found in the same membrane preparation (Lotersztajn, S., Hanoune, J. and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215; Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020). When the entire reconstituted vesicle population was treated with ATP and 45Ca in a buffer containing oxalate, the vesicles with Ca2+ transport activity could be separated from other vesicles by centrifugation in a density gradient and the ATP-dependent Ca2+ transport component was purified approximately 9-fold. This indicates that transport-specific fractionation may be used to isolate the ATP-dependent Ca2+ transport component from liver plasma membrane.

Adenosine Triphosphate↗

PubMed1986

Inhibitory effect of sodium arsenite and azide on asialoglycoprotein receptor mediated endocytosis in suspended rat hepatocytes.

The inhibitory effect of sodium arsenite and azide on asialoorosomucoid endocytosis was tested using isolated rat hepatocytes. Under either continuous flux conditions or a single synchronous wave of ligand endocytosis we confirm that azide inhibits the recycling of the receptors and we provide evidence for the involvement of thiol groups in the internalization step. In addition pretreatment of hepatocytes with azide allows us to demonstrate that receptor endocytosis proceeds independently of the presence of any specific ligand.

Animals↗

PubMed2006

Physical, chemical and environmental properties of selected chemical alternatives for the pre-plant use of methyl bromide as soil fumigant.

Production and use of methyl bromide, a soil fumigant, are being restricted because of this chemical's deleterious effects on stratospheric ozone concentrations. Several products, some of which are currently used as soil fumigants, are being considered as possible replacements for methyl bromide, alone and in various combinations. Among these, 1,3-dichloropropene, methyl isothiocyanate generators such as metam-sodium, and chloropicrin are currently registered, while others such as methyl iodide and sodium azide are at different stages of the registration process. This review examines physicochemical properties, environmental fate, and metabolism of the various potential methyl bromide replacement products.

Agriculture↗

PubMed1988

Mutagenicity, comutagenicity, and antimutagenicity of erythrosine (FD and C red 3), a food dye, in the Ames/Salmonella assay.

Erythrosine (diNa, tetraiodofluorescein) was nonmutagenic to the Ames/Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA104, to a concentration of 2 mg/plate. No mutative intermediates were detected on metabolism by rat caecal cell-free extracts or rat liver S9 mixture; or on incubation with the comutagens, harman and norharman (+/- S9). Instead, an unexpected dose-dependent suppression in spontaneous reversion frequencies was observed (maximum approximately equal to 35% decrease). Erythrosine was antimutagenic to benzo[a]pyrene, but it did not decrease the mutagenicity of the other adduct-forming mutagen, 4-nitroquinoline N-oxide. The food dye was strongly antimutagenic to the bifunctional alkylating agent, mitomycin C, though it did not exhibit a similar effect on the mutagenicity of the corresponding monofunctional agent, methyl methanesulphonate. It partially depressed the mutagenic potentials of sodium azide. The antimutagenic effect of erythrosine on an intercalating agent, ethidium bromide, was discernible only at the highest dose (2 mg/plate). These results have been interpreted in terms of a genointeractive role of erythrosine. Erythrosine produced differential toxic effects in repair-deficient (TA97a, TA98, TA100) and repair-proficient (TA102, TA104) Salmonella tester strains; survival of the repair-deficient strains was found to be decreased. Photoinduced potentiation of erythrosine toxicity was observed, although light irradiation in the presence of erythrosine did not modify the reversion frequencies of the tester strains. The evidence strongly suggests that erythrosine, which exhibits nonmutagenicity in the Ames/Salmonella test, can interact with DNA repair enzymes and/or with DNA.

4-Nitroquinoline-1-oxide↗

PubMed1993

Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes.

Cell killing, oxygen consumption, and hydroperoxide formation were determined in rat hepatocytes after glycolytic and respiratory inhibition. These conditions model the ATP depletion and reductive stress of anoxia ("chemical hypoxia"). Glycolysis was inhibited with iodoacetate, and mitochondrial electron transfer was blocked with sodium azide, cyanide, or myxothiazol. Cell killing, hydroperoxide formation, and inhibitor-insensitive oxygen consumption were greater after azide than after myxothiazol or cyanide. Desferrioxamine, an inhibitor of iron-catalyzed hydroxyl radical formation, delayed cell killing after each of the respiratory inhibitors. Anoxia also delayed cell killing during chemical hypoxia. However, during anoxic incubations, desferrioxamine did not delay the onset of cell death. These findings indicate that reactive oxygen species participate in lethal cell injury during chemical hypoxia. In isolated mitochondria, previous studies have shown that myxothiazol inhibits Q cycle-mediated ubisemiquinone formation in complex III (ubiquinol-cytochrome c oxidoreductase) and that ubisemiquinone can react with molecular oxygen to form superoxide. Decreased killing of hepatocytes with myxothiazol compared with azide suggests, therefore, that mitochondrial oxygen radical formation by complex III is involved in cell killing during reductive stress. In support of this hypothesis, myxothiazol reduced rates of cell killing and hydroperoxide formation in hepatocytes incubated with azide or cyanide. This mitochondrial mechanism for oxygen radical formation may be important in relative but not absolute hypoxia.

Animals↗

PubMed1996

Activation of physiological cell death mechanisms by a necrosis-causing agent.

Cell death is an important physiological process, but it can be triggered by both physiological and nonphysiological stimuli. The product of the bcl-2 gene has the ability to inhibit a physiological cell death process that can be activated by a variety of physiological signals, such as growth factor deprivation. This report describes the use of electron microscopy to examine the effects of two cytotoxic drugs on factor-dependent cells that constitutively express the human bcl-2 gene. Although all cells treated with sodium azide showed changes typical of necrosis, in the absence of Bcl-2 the cells died more rapidly and also displayed features of apoptosis. The fact that Bcl-2 could delay cell death argues that cells can activate internal cell death mechanisms to commit suicide before they are killed by a cytotoxin. Northern analysis showed that growth factor did not preserve viability of the cells through induction of bcl-2. However, growth factor may prevent activation of the physiological cell death mechanisms that bcl-2 can control. This process may constitute a primitive defense response, and blocking it may provide a means of limiting damage caused by otherwise sublethal injuries.

Animals↗

PubMed1994

The acid/base catalyst in the exoglucanase/xylanase from Cellulomonas fimi is glutamic acid 127: evidence from detailed kinetic studies of mutants.

The exoglucanase/xylanase Cex from Cellulomonas fimi hydrolyzes beta-1,4-glycosidic bonds with net retention of anomeric configuration, releasing the disaccharides beta-cellobiose or beta-xylobiose. It uses a double-displacement mechanism involving a glycosyl-enzyme intermediate which is formed and hydrolyzed with general acid/base catalytic assistance. Glu127 was proposed as the acid/base catalyst on the basis of sequence alignments, and mutants at this position were constructed in which the glutamic acid is replaced by alanine or glycine. The following kinetic analysis provides firm support for the assignment of Glu127 as the acid/base catalyst and suggests a more general strategy for identification of this residue in other glycosidases. Substrates which do not require protonic assistance for initial bond cleavage exhibit kcat/Km values similar to those of wild-type enzyme, whereas substrates which do require assistance have kcat/Km values over 6000-fold smaller. Thus rate constants for glycosylation are affected to different degrees by this substitution, depending upon their need for acid catalysis. The deglycosylation rate constant is decreased 200-fold by such substitution, due to the removal of general base catalytic assistance. In the presence of sodium azide a new product, beta-cellobiosyl azide, is formed with these mutants whereas only cellobiose is formed with wild-type enzyme or the Glu127Asp mutant under similar conditions. Addition of azide results in very significant increases in kcat values, ranging from 8-fold for 4''-nitrophenyl cellobioside to over 200-fold for 2'',4''-dinitrophenyl cellobioside, whereas kcat/Km values for these substrates remain essentially constant. No effects on rate upon azide addition are seen with substrates containing aglycons of poor leaving group ability.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinomycetales↗

PubMed1993

Acridine orange-mediated photodamage to cultured cells.

Photosensitization mediated by the lysosomotropic, weakly basic dye acridine orange (AO) was studied on cultured J-774 cells. The phototoxicity was found to be potentiated by elevated oxygen tension and reduced at low oxygen tension. Moreover, cell cultures pre-exposed to the singlet oxygen scavenger sodium azide showed pronounced protection against the loss of viability induced by AO and blue light. AO-mediated photosensitization was neither increased by pre-exposure of cell cultures to ferric chloride or the catalase-inhibitor aminotriazole nor decreased by exposure to deferoxamine. These observations suggest that type II (singlet oxygen-mediated) reactions predominate over type I reactions (radical-mediated). A rapid and pronounced decrease in lysosomal cathepsin L activity (up to 60%) was observed after an initial 10 min irradiation, indicating the lysosomal compartment to be an early target. This irradiation time did not, however, result in any substantial loss of viability. Levels of cytosolic lactate dehydrogenase were unaffected even after 30 min irradiation, indicating that neither cytosol nor plasma membrane is a primary target of the AO-mediated photodamage. Glutathione depletion by pre-exposure to buthionine-S,R-sulfoximine (BSO) much enhanced the sensitivity of J-774 cells to AO-mediated photosensitization, indicating a protective role for thiol-containing compounds against AO-mediated photodamage.

Acridine Orange↗

PubMed1994

Colonic absorption of insulin-like growth factor I in vitro.

Colonic absorption of recombinant human insulin-like growth factor I (rhIGF-I) was measured in vitro using both rat and minipig colon. The permeability coefficients were 8.03 +/- 1.03 and 4.75 +/- 0.43 x 10(-8) cm sec-1 in the rat and minipig, respectively. The steady-state flux in rat colon was linearly related to the donor concentration over the range 1 to 10 mg/mL. rhIGF-I was metabolically stable in contact with both mucosal and serosal surfaces of washed colon for 5 hr. The amount of IGF-I permeating through the tissue was quantitated by radioimmunoassay and the identity and integrity of the permeating species were confirmed by reverse-phase HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting. In all cases the permeant was identical to authentic rhIGF-I. The integrity of the colonic tissue in vitro was demonstrated by the maintenance of electrophysiological parameters, a secretory response to serosal theophylline, and the ability of sodium azide, a metabolic inhibitor, to abolish the barrier properties and cause a large increase in flux.

Animals↗

PubMed1997

Orthovanadate induces cell death in rat dentate gyrus primary culture.

The aim of this study was to define the effects of a potent inhibitor of tyrosine phosphatases, sodium orthovanadate (0.1-100 microM for up to 48 h), on dentate gyrus cells (DGC) in culture. Treatment with 100 microM orthovanadate evoked a delayed form of cell death. To examine the possible involvement of apoptosis in orthovanadate-induced cell death, biochemical and morphological alterations were compared with those of necrotic death induced by sodium azide. Phase-contrast microscopy and nuclear condensation analysis showed that orthovanadate and azide each evoked cell death by distinct pathways. TUNEL assay was positive in both cases. Application of a protein synthesis inhibitor, cycloheximide, did not prevent cytotoxicity caused by either orthovanadate or azide and potentiated the effects of vanadate. We conclude that orthovanadate-induced death of DGC bears features of apoptosis.

Animals↗

PubMed2006

Purification and characterization of Fe-containing superoxide dismutase from Methanobrevibacter arboriphilus strain AZ.

Superoxide dismutase (SOD) was purified from cells of the strict anaerobic methanogenic archaeon Methanobrevibacter arboriphilus strain AZ. The four-step purification procedure resulted in enzyme with specific activity of 3970 units/mg and yield of 22%. It was shown that the SOD is a Fe-containing homotetramer composed of subunits of 21.2 kD each. Sodium azide (13.5 mM), unlike KCN, inhibits the activity of the SOD. Hydrogen peroxide (0.5 mM) inactivates the enzyme, which is consistent with the properties of the known Fe-containing SODs from methanogenic Archaea.

Hydrogen Peroxide↗

PubMed1986

Phenotypic alterations in tumors that developed from threshold subcutaneous inocula. I. Reduced binding of natural antibodies and sensitivity to hypotonic lysis.

Tumors obtained from the injection site of threshold subcutaneous inocula of L5178Y-F9 or SL2-5 lymphomas in syngeneic mice exhibited increased tumor frequencies and reduced sensitivities to other parameters of natural immune resistance in vivo and in vitro. An examination of the resistant phenotype of cells derived through this model of tumor progression revealed that the more aggressive in vivo grown cells were less able to inhibit natural killer (NK) cell cytolysis and to bind natural antibodies (NAb) measured through fluorescence analysis, although they could not be distinguished from the starting clones by absorption of NAb for complement-mediated lysis. The in vivo grown cells also exhibited a reduced sensitivity to hypotonic lysis, which was not detectable after preincubation at 4 degrees C or upon exposure to sodium azide, procedures that reduced the lysis of the starting clones. The differential susceptibility of the in vivo grown cells was increased to control levels by treatment with cycloheximide or colchicine. These studies suggest that a decreased sensitivity to lysis associated with a reduced autolytic process and an increased counterlytic mechanism, in addition to a reduced antigen expression for binding of NK cells and certain NAb contribute to this resistant phenotype, which may characterize tumors that arise under the selective pressure of natural resistance mechanisms in the natural course of neoplastic development.

Absorption↗

PubMedDate not supplied

[The photodynamic inactivation of Candida guilliermondii in the presence of photodithazine].

Photodithazine, a glucosamine salt of chlorin e6, enhanced the inactivation of Candida guilliermondii cells by visible light. The sensitizing effect of photodithazine was found to be related to free or cell surface-bound molecules of this dye. Sodium azide (a singlet oxygen quencher) and propyl gallate (an inhibitor of lipid peroxidation) protected yeast cells from the photodithazine-enhanced photoinactivation.

Antioxidants↗

PubMed1996

Cytochrome f encoded by the chloroplast genome is imported into thylakoids via the SecA-dependent pathway.

In vitro import of the precursor of tobacco cytochrome f, which is is encoded by the chloroplast genome, into isolated pea thylakoids was analyzed. Upon incubation with thylakoids and a stromal fraction, the precursor of cytochrome f was efficiently imported into thylakoids. The imported cytochrome f was tightly integrated into the thylakoid membrane. Insertion of cytochrome f into the thylakoid membrane was blocked by nigericin, sodium azide, and antibodies against pea chloroplast SecA. These results suggest that cytochrome f utilizes the bacterial-type SecA-dependent pathway to be imported into thylakoids.

Adenosine Triphosphatases↗

PubMed1997

Studies on the aerobic photooxidation of cysteine using riboflavin as a sensitizer: evidence for the photogeneration of a superoxide anion and hydrogen peroxide.

The riboflavin sensitized oxidation of cysteine under an aerobic condition was investigated. The effects of various scavengers, such as superoxide dismutase, catalase, mannitol, sodium azide and potassium ferrocyanide (an electron donor), on the photooxidation were determined. A reaction mechanism involving the superoxide anion is proposed for the photooxidation of cysteine to cysteic acid.

Cysteine↗

PubMed2005

Binding and photocleavage of cationic porphyrin-phenylpiperazine hybrids to DNA.

The binding properties of cationic porphyrin-phenylpiperazine hybrids to calf thymus (CT) DNA were investigated by using absorption, fluorescence and circular dichroism (CD) spectra, and the apparent affinity binding constants (K(app)) of the porphyrins for CT DNA were determined by using a competition method with ethidium bromide (EB). Intercalation of porphyrin into CT DNA occurred when two phenylpiperazines were introduced at cis position onto the periphery of cationic porphyrin. The photocleavages of pBR322 plasmid DNA by the porphyrins were consistent with the values of K(app). With [porphyrin]/[DNA base pairs] ratio increased, the binding mode tended to be outside binding, and the cleavage abilities of the porphyrins varied. In the presence of sodium azide, a quencher of 1O2, the cleavage of DNA by the porphyrin of intercalation was less inhibited.

Animals↗

PubMed2002

Detailed kinetic analysis and identification of the nucleophile in alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase.

alpha-l-Arabinofuranosidases cleave the l-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall. The alpha-l-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-alpha-l-arabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme. The steady-state constants and the resulting Brønsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue. The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution. The resulting E294A mutant, with 4-nitrophenyl alpha-l-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K(m) value compared with the wild type enzyme. Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2',4',6'-trichlorophenyl alpha-l-arabinofuranoside by the E294A mutant. The glycosyl-azide product formed during this reaction was isolated and characterized as beta-l-arabinofuranosyl-azide by (1)H NMR, (13)C NMR, mass spectrometry, and Fourier transform infrared analysis. The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the alpha-l-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.

Alanine↗

PubMedDate not supplied

Rapid determination of rice seed vigour by spontaneous chemiluminescence and singlet oxygen generation during early imbibition.

Using a highly sensitive single photon counter, a spontaneous chemiluminescence (CL) study on rice (Oryza sativa L.) seeds stored in different years was carried out. We first observed that the degree of ageing in rice seeds was related to the intensity of spontaneous CL during early imbibition (0-30 min). Rice seeds stored for a shorter time had a stronger intensity of CL in early imbibition. The germination rate of rice seeds showed an obvious positive correlation with the intensity of spontaneous CL. Singlet oxygen ((1)O(2)) in rice seeds during early imbibition was investigated by a CL method using a cypridina luciferin analogue, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2alpha] pyrazin-3-one (MCLA), as a selective CL probe. Additional experimental evidence for the formation of (1)O(2) came from the quenching effect of sodium azide (NaN(3)) on MCLA-mediated rice seeds' CL. Analysis based on the experimental results demonstrated that spontaneous CL in rice seeds during early imbibition was mainly contributed by singlet oxygen ((1)O(2)).

Germination↗