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Results for “Sodium Azide”

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PubMed1991

Sodium nitrite-stimulated metabolic activation of benzo[a]pyrene 7,8-dihydrodiol in human polymorphonuclear leukocytes.

Sodium nitrite was shown to enhance the metabolism of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) to 7/8,9,10- and 7,10/8,9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (tetraols) in phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes (PMNs). The production of these tetraols implicates the intermediate formation of the corresponding trans-7,8-dihydroxy-9,10-epoxy-7,8-9,10-tetrahydrobenzo[a]pyrene (anti-BPDE). A 2- to 3-fold increase in the tetraol yield was observed in the presence of nitrite in excess of 1 mM. Sodium azide, an inhibitor of myeloperoxidase and catalase, reduced the nitrite-stimulated metabolism of BP-7,8-diol in PMA-activated leukocytes. Diphenylene iodonium sulphate, a NADPH-oxidase inhibitor, lowered the production of tetraols in PMA-stimulated leukocytes both in the absence and presence of nitrite. Additionally, nitrite markedly enhanced the covalent binding of metabolites derived from [3H](-)-BP-7,8-diol to leukocyte proteins as well as to DNA present extracellularly. The nitrite-stimulated covalent binding to both proteins and DNA was inhibited by the presence of sodium azide. The mechanism underlying the effect of nitrite on the metabolism of BP-7,8-diol to reactive intermediates in PMA-activated human polymorphonuclear leukocytes is not known. However, the results are compatible with a peroxidase-dependent mechanism although other possible pathways may contribute to the enhanced rate of metabolism.

Biotransformation↗

PubMed1993

The influence of culture conditions on the susceptibility of Salmonella typhimurium in the Salmonella mutagenicity test.

To determine the variability of tester strain susceptibility in the Salmonella mutagenicity assay and to optimize the culturing procedure we examined the influences of the overnight culture period (12-16 h), the use of an additional short-term culturing procedure (1-4 h) and the Salmonella density (cfu/plate) on the rate of revertants per plate. As tester strains we used Salmonella typhimurium TA97, TA98, TA100 and TA102. As shown previously for other microbial genotoxicity short-term tests (i.e. with Escherichia coli PQ37), we observed the highest susceptibility of the Salmonella strains, i.e. the highest amounts of revertants per plate, when using a 12 h overnight culture followed by a 2 h short-term culturing procedure. We calibrated the bacterial count to 100 x 10(6) cfu per assay by photometric measurement (600 nm). Initially, we used 0-1 nmole 2,4,7-trinitro-9-fluorenone with S. typhimurium TA97, 0-15 nmole daunomycin with strain TA98, 0-25 nmole sodium azide with strain TA100 and 0-15 nmole methylmethanesulfonate with strain TA102 as reference compounds in the standard plate incorporation test. Subsequently, to evaluate the results of the culturing procedure variations we examined 22 well-known mutagenic and direct-acting (-S9-mix) compounds out of different chemical classes using both the standard and a modified culturing procedure. The comparison of these results showed a 64% (for 4-nitroquinoline-N-oxide) to 421% (for sodium azide) increased amount of revertants for the modified test protocol.

Colony Count, Microbial↗

PubMed1977

Cyanide-insensitive respiration in Schizophyllum commune.

Two mutants of the hymenomycete Schizophyllum commune, unable to use acetate as the sole carbon source for growth, were isolated. Growth of the mutants on a glucose minimal medium was only slightly inhibited by sodium azide. Genetic analysis revealed mutations in different chromosomal genes in the respective mutants. Both these mutants exhibited a high cyanide-insensitive endogenous respiration. The inhibition of the respiration by 8-hydroxyquinoline showed the mutants to respire predominantly by an alternative respiratory pathway observed in many fungal species, but not in the hymenomycetes so far. An enhanced cyanide-insensitive respiration was also found in a wild-type strain of Schizophyllum commune grown in the presence of sodium azide.

Acetates↗

PubMed1990

Microphotometric determination of enzymes in brain sections. II. GABA transaminase.

The tetrazolium salt procedure of van Gelder (1965) for the demonstration of GABA transaminase (GABAT; the most important GABA degrading enzyme) was adapted for microphotometric measurements of GABAT activities in brain sections using the hippocampus of rats as selected brain region. The final incubation medium consisted of 50 mM GABA, 5 mM alpha-ketoglutarate, 7 mM NAD, 10 mM sodium azide, 6 mM nitroblue tetrazolium chloride, 20 mM malonate and 15% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 8.0. There was a linear relationship between GABAT activity and section thickness up to 14 microns and between GABAT activity and reaction time at least up to 20 min (kinetic and end-point measurements). Phenazine methosulfate as an exogenous electron carrier and pyridoxal-5-phosphate as coenzyme of GABAT did not enhance the demonstrable GABAT activities, whereas sodium azide as a blocker of the respiratory chain resulted in an increase of demonstrable enzyme activities. A coreaction of succinate dehydrogenase was excluded by the use of malonate (competitive inhibitor). Using the incubation medium described GABAT activities were demonstrated via the endogenous enzymes succinic semialdehyde dehydrogenase and NADH tetrazolium reductase which were shown to be not rate limiting and seems to be similarly localized as GABAT.

4-Aminobutyrate Transaminase↗

PubMed1979

Capping of surface immunoglobulin on 'hairy cells' is independent of energy production.

Capping, independent of metabolic energy, of surface immunoglobulin (S-Ig) on 'Hairy cells' from patients with Hairy cell leukaemia (HCL) is described. As controls leukaemic cells from a patient with a prolymphocytic leukaemia (PLL) and blood lymphocytes from healthy individuals were used. The specificity of the energy-independent capping of the HCL-cells as compared to the controls was tested by incubation of the cells at 4 degrees C in the presence of 0.1 M sodium azide with different FITC-labelled ligands. In order to find an explanation for this phenomenon, the influence of cytochalasin B, colchicine and the combination of both drugs on capping of S-Ig and concanavalin (Con-A)-receptors at 37 degrees C was investigated. Furthermore the effect of Con A on S-Ig capping and vice versa was studied. The results show that only S-Ig on HCL cells could form caps at 4 degrees C in the presence of sodium-azide. Cytochalasin B alone induced a strong inhibition of Con A capping on all 3 cell types, whereas S-Ig capping was unaffected. Colchicine alone had practically no effect. Anti-Ig inhibited subsequent patch and cap formation with Con A on both HCL cells and PLL cells, whereas Con A caps and patches were redistributed by anti-Ig on PLL cells, but not on HCL cells. Conversely, Con A could link S-Ig to other receptors, leading to inhibition of S-Ig capping at 4 degrees C on HCL cells and to co-capping of S-Ig at 37 degrees C on both cell types. In addition Con A induced redistribution of S-Ig caps. The combination of co-capping of S-Ig by Con A, followed by redistribution of the caps by FITC-anti-Ig simulated inhibition of S-Ig capping by Con A on PLL cells. The major conclusions are: in some cases inhibition of capping may actually be caused by redistribution of caps; the energy-independent capping cannot be explained by free diffusion of S-Ig in the membrane through lack of any connexion with receptor-mobility regulating systems. It is proposed that the energy requirement of capping is needed to inactivate a specific mechanism,w which restrains receptor mobility and which is non-operative in HCL cells.

Adult↗

PubMed1991

Assessment of mitochondrial membrane potential as an indicator of cytotoxicity.

Mechanistically based short-term in vitro tests to evaluate the relative cytotoxicity of of chemicals will complement in vitro genotoxicity testing during the initial phases of toxicity evaluation as well as provide information on the cellular site of action for chemicals found to be toxic in animals. The objective of this study was to characterize a procedure for evaluating mitochondrial membrane potential, an integral component of cellular energy homeostasis and normal cellular function, as an in vitro indicator of chemically induced cytotoxicity. Rhodamine 123, a cationic fluorescent dye whose mitochondrial fluorescence intensity decreases quantitatively in response to dissipation of mitochondrial transmembrane potential, was used to evaluate disturbances in mitochondrial membrane potential. Cultured rat liver epithelial cells (WB cell line) or human skin fibroblasts (MSU-2 cell line) treated with the oxidative phosphorylation uncoupler 2,4-dinitrophenol (DNP) or the cytochrome oxidase inhibitor sodium azide were used to characterize the system. In addition, acetaldehyde, which has been reported to damage the plasma membrane, but not the mitochondrial membrane, was used to demonstrate the specificity of this assay system. Mitochondrial membrane potential was not significantly affected by the cell culture density, as long as the cells were in the logarithmic phase of growth. The stage of the cell cycle influenced the mitochondrial membrane potential in human skin fibroblasts (highest in late G1-early S) but not in rat liver cells. DNP and sodium azide significantly (p less than 0.01) reduced the mitochondrial membrane potential in both cell lines compared to untreated cells, while acetaldehyde did not reduce the mitochondrial membrane potential in either cell line. This assay provides a tool for evaluating the effect of chemical treatments on mitochondrial membrane potential, as well as an indicator of cytotoxicity which does not require the use of animals.

Animals↗

PubMed1982

Effect of p-p'-DDE administered in vivo and in vitro on Ca2+ binding and Ca2+-Mg2+-ATPase activity in egg shell gland mucose of ducks.

Thinning of the egg shell is produced by p-p'-DDT and DDE in several species of birds. A study was made of the effect of DDE administered in vitro and in vivo on the Ca2+ binding and Ca2+-Mg2+-activated ATPase of a homogenate of the egg shell gland of ducks (Anas platyrhynchos var.). The concentration of Ca2+ was 1 X 10(-4) M and that of MgATP 1 X 10(-3) M. In vitro, DDE in concentrations of 2-16 micrograms/ml of incubation medium inhibited the Ca2+-Mg2+-activated ATPase in a concentration-dependent manner, whereas Mg2+-activated ATPase was not affected by these concentrations. The Ca2+ binding by the homogenate was reduced by DDE in the same concentrations. The sodium azide sensitive Ca2+ binding was most sensitive. In vivo, DDE administered in a concentration of 40 mg/kg dry weight of the food for 45 days reduced the egg shell index by 18% in comparison to controls. After 45 days of treatment the DDE concentrations in the egg shell gland mucosa was 1.20 +/- 0.16 micrograms/g of wet weight, while no DDE was detected in the controls. The Ca2+-Mg2+-activated ATPase was reduced by 32%, whereas the Mg2+-ATPase was not changed. The Ca2+ binding by the homogenate was reduced by 29%, the sodium azide sensitive part being most vulnerable, DDE increased the total Ca content of the egg shell gland mucosa by 44%. Since Ca is transported against a concentration gradient between blood plasma, and the lumen of the shell gland, it is suggested that DDE, by inhibiting the Ca2+-Mg2+-activated ATPase, decreased the Ca translocation over the egg shell gland mucosa.

Animals↗

PubMed2006

NO donors inhibit Na,K-ATPase activity by a protein kinase G-dependent mechanism in the nonpigmented ciliary epithelium of the porcine eye.

1. We developed a novel method to isolate nonpigmented epithelial (NPE) cells from porcine eyes in order to examine Na,K-ATPase responses to nitric oxide (NO) donors specifically in the epithelium. 2. Cells were treated with NO donors and other test compounds for 20 min prior to Na,K-ATPase activity measurement. 3. NO donors, sodium nitroprusside (SNP, 1 microM-1 mM), sodium azide (100 nM-1 microM) and S-nitroso-N-acetylpenicillamine (1 microM-1 mM) caused significant concentration-dependent inhibition of Na,K-ATPase activity. Detection of nitrite in the medium of L-arginine and SNP-treated NPE confirmed NO generation. 4. Concentration-dependent inhibition of Na,K-ATPase was also obtained by L-arginine (1-3 mM), a physiological precursor of NO and 8p-CPT-cGMP (1-100 microM), a cell permeable analog of cGMP. The L-arginine effect was abolished when the NO synthesizing enzyme, NO-synthase, was inhibited by L-NAME (100 microM). 5. The inhibitory effect of SNP or sodium azide on Na,K-ATPase activity was suppressed by soluble guanylate cyclase (sGC) inhibitors, ODQ (10 microM) or methylene blue (10 microM). 6. The inhibitory effect of 8p-CPT-cGMP on Na,K-ATPase was abolished by protein kinase G (PKG) inhibitors, H-8 (1 microM) and H-9 (20 microM), but not by the protein kinase A (PKA) inhibitor H-89 (100 nM). H-8 and H-9 partially suppressed the inhibitory effect of SNP on Na,K-ATPase. 7. Taken together the results indicate that Na,K-ATPase inhibition response to NO donors involves activation of sGC, generation of cGMP and activation of PKG. These findings suggest that Na,K-ATPase inhibition in NPE may contribute to the ability of NO donors to reduce aqueous humor secretion.

Animals↗

PubMed2003

Uptake of 5-methyltetrahydrofolate into PC-3 human prostate cancer cells is carrier-mediated.

Uptake of 5-methyltetrahydrofolate into the PC-3 human prostate cancer cells was linear for the first 60 min. There was no difference in the initial rate of uptake in cells incubated in folate-free medium for 24 or 48 hr compared to control cells grown in folate-containing medium. The initial rate of 5-methyltetrahydrofolate uptake showed little dependence on extracellular pH and it was independent of extracellular sodium ions. Transport of 5-methyltetrahydrofolate into PC-3 cells was saturable - K(m) = 0.74 micro M and V(max) = 7.78 nmol/10(9)cells/min and these kinetic constants were not different in cells incubated for 24 hr in folate-free medium (K(m) = 0.80 +/- 0.22, V(max) = 8.52 +/- 0.50; P = 0.09, N = 3). Uptake of 5-methyltetrahydrofolate was inhibited by structural analogs with the K(i) values being 0.50, 1.79, and 31.8 micro M for 5-formyltetrahydrofolate, methotrexate, and folic acid, respectively. Uptake of 5-methyltetrahydrofolate was inhibited by the energy poisons, sodium cyanide, sodium arsenate, p-chloromercuriphenylsulfonate, and sodium azide. Uptake was inhibited by increasing concentrations of sulfate and phosphate ions, suggesting that 5-methyltetrahydrofolate may be transported by an anion-exchange mechanism. These results show that 5-methyltetrahydrofolate is transported into PC-3 prostate cancer cells by a carrier-mediated process.

Arsenates↗

PubMed1994

Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a specific endothelial cell mitogen with potent angiogenic properties. In tumors, VEGF has been localized to the most necrotic and ischemic areas of the tissues, suggesting that local hypoxia is a potent inducer of VEGF production. Initial experiments in vitro confirmed the stimulatory effect of hypoxia on VEGF expression. The extent of this response and the mechanisms involved in oxygen sensing are poorly characterized. EXPERIMENTAL DESIGN: Confluent monolayers of malignant cell lines or primary cultures of fibroblast or endothelial cells were exposed to hypoxia or incubated with either cobalt chloride, a stimulator of erythropoietin gene expression, or sodium azide, an inhibitor of oxydative phosphorylation. VEGF expression was analyzed by Northern blot or RNase protection assays. The expression VEGF in vivo was studied in animals subjected to hypobaric hypoxia or functional anemia. RESULTS: Hypoxia greatly stimulated VEGF expression in tumor cell lines and primary fibroblast cultures. Endothelial cells, that expressed very low constitutive levels of VEGF, were resistant to hypoxic stimulation. RNase protection analysis showed that hypoxia primarily stimulated the induction of smaller and medium VEGF isoforms, i.e., the same ones expressed under normal conditions. The stimulatory effect of hypoxia on VEGF could be reproduced in vitro by cobalt chloride but not with sodium azide. In vivo, both hypoxia and anemia were found to be potent inducers of VEGF expression in several organs including heart, brain, liver, kidney, and muscle. As in vitro, cobalt was also found to be a potent stimulator of VEGF in vivo. CONCLUSIONS: Hypoxia is a potent inducer of VEGF expression in malignant as well as normal cultured cells. It is also a stimulator of VEGF expression in vivo. The VEGF gene appears to respond to hypoxia like the erythropoietin gene, and the mechanism of oxygen sensing probably is mediated by a heme-containing protein.

Anemia↗

PubMed2000

Effect of vanillin on methylene blue plus light-induced single-strand breaks in plasmid pBR322 DNA.

The ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, in inhibiting photosensitization-induced single-strand breaks (ssbs) in plasmid pBR322 DNA has been examined in an in vitro system, independent of DNA repair/replication processes. Photosensitization of DNA with methylene blue, visible light and oxygen, induced ssbs resulting in the production of open circular form (OC form) in a concentration-dependent manner. The yield of OC form induced by photosensitization was increased several-fold by deuteration of the buffer and was found to be inhibited by sodium azide, a scavenger of singlet oxygen (1O(2)). Vanillin, per se, did not induce but inhibited photosensitization-induced ssbs in plasmid DNA, at millimolar concentrations. The inhibitory effect of vanillin was both concentration- and time-dependent. On a molar basis, vanillin was, however, less effective than trolox, a water-soluble analogue of alpha-tocopherol. Photosensitization by methylene blue system generates singlet oxygen, as one of the major components of ROS. Therefore, interaction of singlet oxygen with vanillin was investigated. The rate constant of vanillin with 1O(2) was estimated to be 5.93x10(7)M(-1)s(-1) and that of sodium azide as 2. 7x10(8)M(-1)s(-1). The present investigations show that vanillin can protect against photosensitization-induced ssbs in the plasmid pBR322 DNA, and this effect may partly be due to its ability to scavenge 1O(2).

Antioxidants↗

PubMed1999

Reactive oxygen species in essential hypertension and non-insulin-dependent diabetes mellitus.

To evaluate whether increased levels of reactive oxygen species (ROS) are involved in the pathogenesis of essential hypertension (EH) and non-insulin-dependent diabetes mellitus (NIDDM), both resting and stimulated levels of intracellular ROS were measured in lymphocytes from patients with EH (n = 10), NIDDM (n = 16) and age-matched healthy individuals (control subjects, n = 19). ROS was monitored with the dye, dihydrorhodamine-123 (DHR; 1 micromol/L) in the presence or absence of superoxide dismutase (superoxide scavenger), sodium azide (singlet oxygen/hydrogen peroxide scavenger), genistein (tyrosine kinase inhibitor), or bisindolylmaleimide (protein kinase C inhibitor). Simultaneous monitoring of cytosolic [Ca2+]i was done with fura-2. Resting ROS levels were significantly higher in NIDDM (4.71+/-0.25 nmol/10(6) cells; mean +/- SEM, P<.05) compared with EH (4.03+/-0.22 nmol/10(6) cells) or controls (4.05+/-0.15 nmol/10(6) cells). The formyl-Met-Leu-Phenylalanine-(fMLP)-induced ROS generation was significantly higher in NIDDM (21.92+/-2.23 nmol/10(6) cells; P<.05) compared with EH (14.58+/-1.90 nmol/10(6) cells) or control (16.06+/-1.22 nmol/10(6) cells). The fMLP-induced ROS increase was significantly reduced in the presence of sodium azide in all groups (P<.01) but was largely unaffected in the presence of SOD. Genistein and bisindolylmaleimide significantly inhibited the fMLP-induced ROS in all groups. The fMLP-induced [Ca2+]i increase was significantly higher in NIDDM (71+/-12 nmol/L, P <.01) compared with EH (42+/-4 nmol/L) and control subjects (35+/-3 nmol/L). Phytohemagglutinin was more effective in increasing [Ca2+]i than ROS. It is concluded that ROS may play a role in the metabolic syndrome of NIDDM but not in EH.

Biomarkers↗

PubMed1996

Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance.

Fluconazole-susceptible Candida albicans strains accumulated [3H]fluconazole at a rate of approximately 2 pmol/min per 10(9) cells. Fluconazole accumulation was not affected by the pretreatment of cells with sodium azide or with 2-deoxyglucose. The rate of fluconazole accumulation became saturated at high fluconazole concentrations and was not affected by the addition of ketoconazole, and there was no fluconazole accumulation in cells incubated at 4 degrees C. A fluconazole-resistant mutant of C. albicans SGY-243 was isolated following growth enrichment in fluconazole-containing medium. Cells of the mutant strain, designated FR2, showed a reduced rate of fluconazole accumulation compared with SGY-243 and were not resistant to other azole antifungal agents. The rates of fluconazole accumulation by C. albicans FR2 and the other azole-resistant strains, B59630, AD, and KB, were increased in the presence of sodium azide, suggesting that fluconazole resistance in these strains may be associated with an energy-dependent drug efflux. Fluconazole-resistant C. albicans strains all contained elevated amounts (2- to 17-fold) of mRNA encoding Cdr1, and an ATP-binding cassette-type transporter. In addition, C. albicans FR2 also contained increased amounts of mRNA encoding Benr, a major facilitator superfamily transporter. These results suggest that fluconazole enters C. albicans cells by facilitated diffusion and that fluconazole resistance may involve energy-dependent drug efflux associated with increased expression of Benr and/or Cdr1.

Antifungal Agents↗

PubMed1980

Reversible and irreversible loss of Fc receptor function of human monocytes as a consequence of interaction with immunoglobulin G.

The effects of IgG in different configurations on the Fc receptor function of human monocytes were studied. Receptor function was assessed by quantitating immune adherence and/or ingestion of human erythrocytes coated with IgG anti-D antibody. Monomeric IgGl in solution inhibited the Fc receptor function of monocytes, but this function was restored completely after washing. In contrast, monomeric IgG that was adsorbed nonspecifically to a plastic surface inhibited the Fc receptor function of monocytes even after washing away unbound IgGl. This loss of function could be blocked by sodium azide and was reversed when the IgG adsorbed to plastic was degraded by trypsin, suggesting that loss of function was the reversible consequence of localized binding of most of the monocyte's receptors at the point of contact with immobilized IgGl. Fluid-phase aggregates of IgGl also reduced the Fc receptor function of monocytes as a consequence of direct binding to the monocyte surface. High concentrations of purified aggregates rapidly reduced Fc receptor function but function was reversed by trypsin even after incubation for 18 h. Lower concentrations of aggregates reduced Fc receptor function more slowly, but after 18 h of incubation, lost function was not restored by trypsin treatment. Because the transition from reversible to irreversible loss was blocked by sodium azide, an energy-dependent process of ingestion, shedding or denaturation of receptors is responsible for this irreversible loss of Fc receptor function. Rabbit IgG anti-human IgG bound to IgG adsorbed to the surface of monocytes also mediated a loss of Fc receptor function as a result of the binding of Fc receptors to the Fc portion of the rabbit IgG molecule, a process analogous to the binding of aggregated IgG. After irreversible depletion of Fc receptor function by anti-IgG, partial recovery of function was detectable within 12-24 h of incubation in vitro, and this recovery was blocked by cycloheximide, suggesting that new receptor synthesis was required for restoration of function.

Antibodies, Anti-Idiotypic↗

PubMed2003

Sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells.

OBJECTIVE: To study the sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells. METHODS: Sensitivity and specificity of several common methods for apoptosis determination were evaluated (Apo2.7-expression, Annexin V-binding, TUNEL-reaction, poly-(ADP-ribose)-polymerase-(PARP) cleavage and single-stranded-DNA (ssDNA) staining). Apoptosis was induced by oxidative stress generated by hydrogen peroxide in 3 cultured cells types growing as adherent monolayer (MiaPaCa-2, Hep-G2 and human skin fibroblasts), necrosis was induced by depletion of cellular ATP using sodium azide. Cells positively stained by the respective apoptosis assay were quantified and alterations of cell morphology were monitored by fluorescence microscopy. The date was analyzed by one-way analysis of variance and significance test of correlation coefficient. RESULTS: One hour after apoptosis induction significant cell fractions were positively stained for ssDNA (33% with MiaPaCa-2 cells, 35% with Hep-G2 cells, 56% with human skin fibroblasts). PARP-cleavage was less sensitive compared to the ssDNA-staining. Apo2.7-expression, Annexin V-binding and TUNEL-reaction were not applicable to detect early apoptosis induced by oxidative stress (below 2 hours), but were efficiently monitoring late apoptosis. Specificity of ssDNA-staining was complete with each cell type even 4 hs after induction of necrosis by the highest sodium azide concentration. In contrast, the same experimental conditions resulted in 50% - 90% positively stained necrotic cells by using Apo2.7-expression, TUNEL-reaction or Annexin V-binding. Surprisingly, specificity of PARP-cleavage was highly depending on the respective cell type. CONCLUSIONS: Our study prove that among the five methods investigated only ssDNA-staining allowed to completely differentiate apoptosis from necrosis, and is thus suitable to reliably detect early as well as late apoptosis. Therefore, the ssDNA-staining may be used as reference method to clearly identify apoptosis induced by oxidative stress in adherent cells. The TUNEL-reaction, annexin-V-binding and Apo-2.7-expression may be used to quantify the number of apoptotic and necrotic cells especially at later stages but without discrimination of apoptosis and primary or secondary necrosis.

Annexin A5↗

PubMed1971

Characterization of the -lactamase specified by the resistance factor R-1818 in E. coli K12 and other Gram-negative bacteria.

1. The amino acid composition of the beta-lactamase from E. coli (R-1818) was determined. 2. The R-1818 beta-lactamase is inhibited by formaldehyde, hydroxylamine, sodium azide, iodoacetamide, iodine and sodium chloride. 3. The K(m) values for benzylpenicillin, ampicillin and oxacillin have been determined by using the R-factor enzyme from different host species. The same values were obtained, irrespective of the host bacterium. 4. The molecular weight of the enzyme was found to be 44600, and was the same for all host species. 5. The relationship of R-1818 and R-GN238 beta-lactamases is discussed.

Amino Acids↗

PubMed1984

Effects of phenothiazines on inhibition of plasma membrane ATPase and hyperpolarization of cell membranes in the yeast Saccharomyces cerevisiae.

The transmembranal potential, in Saccharomyces cerevisiae, has been calculated from the distribution ratio of the lipophilic cation tetraphenylphosphonium (TPP+) between the intracellular and extracellular water. Trifluoperazine at concentrations of 10 to 50 microM, caused a substantial increase in the membrane potential (negative inside). This increase was observed only in the presence of a metabolic substrate and was eliminated by the addition of the protonophores 2,4-dinitrophenol and sodium azide, removal of glucose, replacement of glucose by the nonmetabolizable analog 3-O-methyl glucose, or by the addition of 100 mM KCl. An increase in 45CaCl2 accumulation from solutions of low concentrations (1 microM) was observed under all conditions where membrane potential was increased. Proton ejection activity was monitored by measurements of the rates of the decrease in the pH of unbuffered cell suspensions in the presence of glucose. Trifluoperazine inhibited the changes in medium pH; this inhibition was not the result of an increase in the permeability of cell membranes to protons since in the absence of glucose, trifluoperazine did not cause a change in the rate of pH change generated by proton influx. The activity of plasma membrane ATPase was measured in crude membrane preparations in the presence of sodium azide to inhibit mitochondrial ATPase. Trifluoperazine strongly inhibited the activity of the plasma membrane ATPase. The effect of phenothiazines on transport and on membrane potential reported in this study and in the previous one (Eilam, Y. (1983) Biochim. Biophys. Acta 733, 242-248) were observed only in the presence of a metabolic substrate. The possibility that energy is required for the uptake of phenothiazines into the cells was eliminated by results showing energy-independent uptake of [3H]chlorpromazine. The results strongly suggest that phenothiazines activate energy-dependent K+-extrusion pumps, which lead to increased membrane potential. Increased influx of calcium seems to be energized by membrane potential, and therefore stimulated under all conditions where membrane potential is increased. The analog which does not bind to calmodulin, trifluoperazine sulfoxide, had no effect on the cells, but the involvement of calmodulin in the processes altered by trifluoperazine cannot as yet, be determined.

Adenosine Triphosphatases↗

PubMed1983

Role of myeloperoxidase in the respiratory burst of human neutrophils.

Myeloperoxidase (MPO), a heme enzyme present in the primary granules of polymorphonuclear leukocytes (PMNs), has been demonstrated to participate in the oxygen-dependent microbicidal activity of these cells. Evidence for the importance of MPO in this role comes in part from studies of normal PMNs treated with the heme enzyme inhibitor, sodium azide. MPO has also been suggested to regulate the respiratory activity of PMNs during phagocytosis. The role of MPO in PMN oxygen metabolism was examined by studying parameters of the respiratory burst of PMNs from a number of unrelated MPO-deficient subjects; in addition, the ability of heme enzyme inhibitors to duplicate the MPO-deficient state was studied by treating normal and MPO-deficient cells with these compounds. MPO-deficient PMNs were found to have a time-dependent hypermetabolic response as assessed by measurement of oxygen consumption, superoxide generation, hydrogen peroxide release, and hexose monophosphate shunt activity. Catabolic pathways for hydrogen peroxide were normal, suggesting the increased recovery of oxygen metabolites reflects increased production rather than decreased catabolism of H2O2. These observations support the concept that MPO may play an important role in terminating the respiratory burst of normal PMNs. The three heme enzyme inhibitors studied--sodium azide, potassium cyanide, and 3-aminotriazole--differed greatly in the degree to which they inhibited various enzymatic systems in the PMN. Nonetheless, as a group, they exerted qualitatively similar effects on oxygen metabolism of normal and of MPO-deficient PMNs. This indicates that many of the mechanisms by which heme enzyme inhibitors influence PMN metabolism are independent of the inhibition of MPO. Conclusions from studies using such treatment of PMNs should be interpreted with caution.

Catalase↗