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PubMed1997

Airborne particulate matter modulates the production of reactive oxygen species in human polymorphonuclear granulocytes.

Causal relationships between airborne particles (especially particulate matter < 10 microm in diameter) and increases in prevalences and symptoms of respiratory diseases have been postulated in many epidemiologic studies. Polymorphonuclear leukocytes (PMN) in the nasal or bronchial epithelium can be exposed to particulate matter (PM) and may upon exposure produce reactive oxygen species (ROS). Release of ROS can result in cellular and tissue damage and initiate or exacerbate inflammation. To elucidate the effect of PM on inflammatory reactions, we exposed human PMN to PM extracts. PM were collected with high volume samplers in two cities, Dusseldorf and Duisburg, in Germany and reflect sites with high traffic and industrial emissions respectively. The collected particles were extracted using water and then dichloromethane, resulting in an aqueous and an organic extract. The production of ROS was determined using luminol-enhanced chemiluminescence (LCL) of resting and zymosan-stimulated PMN. The present study shows that extracts of PM alone significantly stimulated the production and release of ROS in resting PMN. The effects of the PM extracts were inhibited by superoxide dismutase (SOD), catalase and sodium azide (NaN3). Zymosan-induced LCL was, however, diminished by coincubation with PM extracts. The chemical composition is important when considering the effects of particles. Our study shows that only organic substances adsorbed to particles stimulate LCL. SOZ-induced LCL is inhibited by both types of extracts, but aqueous extracts have a stronger inhibitory effect. It is at present unclear which substances are responsible for these effects.

Air Pollutants↗

PubMed2005

Identification and characterization of a Na+-dependent neutral amino acid transporter, ASCT1, in rabbit corneal epithelial cell culture and rabbit cornea.

PURPOSE: The aim of this study was to investigate the presence of a Na+-dependent neutral amino acid transporter, ASCT1, in rabbit primary corneal epithelial cell culture and rabbit cornea. METHODS: Uptake studies were carried out on rabbit primary corneal epithelial culture (rPCEC) cells using 12-well plates. Transport studies were conducted with isolated rabbit corneas at 34 degrees C. Uptake and transport of L-alanine was determined at various concentrations. Inhibition studies were conducted in presence of various L- and D-amino acids, metabolic inhibitors like ouabain and sodium azide, and in the absence of sodium to delineate the functional characteristics of L-alanine uptake and transport. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA harvested from rabbit cornea and rPCEC cells for identification of ASCT1. RESULTS: Uptake of L-Ala was found to be saturable with a Km of 0.71 mM and a Vmax value of 0.84 micromoles min(-1) mg(-1) protein. Uptake was independent of pH and energy but depends on sodium. It was inhibited by serine, threonine, cysteine, and glutamine but did not respond to BCH (2-aminobicyclo [2,2,1] heptane-2-carboxylic acid) and MeAIB (alpha -methylaminoisobutyric acid). Transport of L-Ala across rabbit cornea was also saturable (Km 6.52 mM and Vmax 1.09 x 10(-2) micromoles min(-1) cm(-2)), energy independent, and subject to similar competitive inhibition. Presence of ASCT1 on rPCEC and on rabbit cornea was identified by RT-PCR. CONCLUSIONS: L-Alanine, the chosen model substrate, was actively transported by Na+-dependent, neutral amino acid exchanger ASCT1, which was identified and functionally characterized on rPCEC cells and rabbit cornea.

Alanine↗

PubMed1982

Preliminary studies of the metabolic activity of purified suspensions of Mycobacterium leprae.

Mycobacterium leprae isolated from armadillo tissue incorporated radioactivity from D-(14C)glucose and (14C)protein hydrolysate. In the presence of glucose, the rate of incorporation of (14C)protein hydrolysate was increased. Uptake of glucose was inhibited by 2-deoxy-D-glucose and sodium azide; that of the amino acids was inhibited by puromycin and chloramphenicol and, weakly, by cycloheximide.

Amino Acids↗

PubMed1994

Characterization and modulation of drug transport kinetics in K562 c1.6 daunorubicin-resistant cell line.

The effects of cyclosporin A (CSA) and cellular energy depletion on daunorubicin (DAU) transport kinetics were investigated in a human erythroid leukaemia cell line K562 c1.6 selected for resistance to daunorubicin. K562 c1.6/DAU resistant cells displayed high levels of P-glycoprotein and a high level of multidrug resistance against several antitumour drugs. The resistance factors of K562 c1.6/DAU cells to DAU, doxorubicin, vinblastine and etoposide were 106, 114, 85 and 13 respectively. A 1.6-fold decrease (P < 0.01, n = 8) in DAU accumulation and a 4-fold increase (P < 0.001, n = 8) in DAU efflux were shown in the resistant cells when compared to K562 c1.6 drug-sensitive parental cells. K562 c1.6/DAU cells were also shown to reach a DAU saturation level (SL) 8-fold faster (P < 0.001, n = 8) than the parental cells. Addition of CSA to the resistant cells led to a dose-dependent increase in cellular DAU retention, while no such effect was observed in the sensitive cells by the introduction of CSA. Resistance to the antitumour drugs could be reduced to various extents by CSA. The patterns of changes and modulations of DAU transport kinetics, as well as chemosensitivity in K562 c1.6/DAU cells were found to be similar to a vinblastine-resistant leukaemia cell line CEM/VLB100. However, K562 c1.6/DAU cells were more resistant to DAU, doxorubicin and etoposide than the CEM/VLB100 cells. An increase in DAU accumulation, intracellular SL and the time to reach 90% saturation level (SL90), and a decrease in DAU efflux in the resistant but not the sensitive cells were found in response to ATP depletion by sodium azide. These effects could be completely reversed by addition of glucose. Our results suggest that the presence of an energy-dependent effluxing mechanism responsible for the decreased drug accumulation and enhanced drug efflux may make a major contribution to the mechanism of resistance in K562 c1.6/DAU resistant cells.

Antineoplastic Agents↗

PubMedDate not supplied

A novel mitochondrial signaling pathway activated by visible-to-near infrared radiation.

The number of cells attached to glass substratum increases if HeLa cell suspension is irradiated with monochromatic visible-to-near infrared radiation before plating (the action spectrum with maxima at 619, 657, 675, 700, 740, 760, 800, 820, 840 and 860 nm). Treating of cell suspension with sodium azide (2 x 10(-5) M), sodium nitroprusside (5 x 10(-5) M), ouabain (1 x 10(-6) M) or amiloride (1.7 x 10(-5) M) before irradiation significantly modifies the spectrum of cell attachment enhancement. A light-induced mitochondrial signaling pathway can be regulated by small ligands directly binding to the catalytic center of cytochrome c oxidase (N(3), NO) as well as by chemicals specifically binding to plasma membrane enzymes (ouabain, amiloride). The comparative analysis of action spectra allows the conclusions that first, Cu(A) and Cu(B) chromophores of cytochrome c oxidase could be involved as photoacceptors and second, various signaling pathways (reaction channels) between cytochrome c oxidase and cell attachment regulation are at work.

Cell Adhesion↗

PubMed1994

The ambivalent role of glutathione in the protection of DNA against singlet oxygen.

Glutathione (GSH) was examined with respect to its ability to protect DNA against 1O2 damage. We have found that GSH protected, at least partly, the DNA against inactivation by 1O2. Up to 10 mM the protection increased as a function of GSH concentration. Above 10 mM the protection remained constant and less than expected on the basis of scavenging/quenching of 1O2, in contrast to the protection offered by sodium-azide. Especially at the higher concentrations of GSH the protection against the biological inactivation is accompanied by an increase in single-strand breaks and also probably lethal base damage. However, all together the data suggest that at least in the physiologically important range (0.1-10 mM) GSH is able to protect efficiently against 1O2-induced inactivating DNA damage.

Azides↗

PubMed1992

Antimutagenic activity of plumbagin in Ames Salmonella typhimurium test.

This paper reports the antimutagenic activity of plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) against certain known chemical mutagens in a standard mutagenicity test system of Ames using S. typhimurium strains. Plumbagin by itself did not show any mutagenic effect, whereas it reduced significantly the mutagenic effect of 4-nitrophenylene diammine, phenyl hydrazine and sodium azide in test strains of S. typhimurium, suggesting that plumbagin possessed antimutagenic activity.

Azides↗

PubMed1983

Production of H2O2 by bovine blood and milk polymorphonuclear leucocytes.

The ability of bovine polymorphonuclear leucocytes (PMN) to release H2O2 was investigated. Resting PMN suspended in buffer released only small amounts of H2O2 which was appreciably increased during phagocytosis of heat killed coliforms. However, in the presence of bovine serum (BS), foetal calf serum (FCS) and milk whey (MW) no increase of H2O2 could be detected unless sodium azide (NaN2) was added. It appears that the enzyme content of these fluids (catalase and lactoperoxidase) consumed the released H2O2 and NaN2, which inactivates these enzymes, abolished this interference. Live organisms required BS or MW both for phagocytosis and for H2O2 production. Bovine IgG2 and, to a lesser extent, IgG1 but not SIgA or IgM stimulated the release of H2O2 independently of phagocytosis; this indicates the presence of receptors specific for IgG2 and IgG1 on the cell surface. Ingestion of casein micelles triggered the greatest burst of H2O2 production by cells suspended in buffer. In general, PMN isolated from blood were more active than cells isolated from milk. Since the extracellular release of H2O2 reflects the intracellular level of H2O2, the lower metabolic activity of milk PMN may contribute to the lesser intracellular bactericidal activity of milk leucocytes. The possibility that the release of H2O2 may activate extracellularly the lactoperoxidase system, known to be bactericidal in milk, is discussed.

Animals↗

PubMed1997

The influence of some compounds commonly used in biochemical studies of mitochondria on the activity of monoamine oxidases.

It was previously described that low concentrations of sodium azide monoamine oxidase (MAO) B assayed by spectrophotometric measurement of benzaldehyde or by hydrogen peroxide accumulation. We failed to confirm this effect using radiometric determination of MAO activity. Tris or dinitrophenol inhibit MAO. The data suggest that some "regulatory effects" depend on the assay of MAO activity.

2,4-Dinitrophenol↗

PubMed2004

Angiotensin type 2 receptor neuroprotection against chemical hypoxia is dependent on the delayed rectifier K+ channel, Na+/Ca2+ exchanger and Na+/K+ ATPase in primary cortical cultures.

We have previously reported that angiotensin II (Ang II) protects cortical neurons from chemical-induced hypoxia through activation of the angiotensin type 2 (AT(2)) receptor. Here, we show in mouse primary neuronal cultures that the AT(2) receptor neuroprotection results from the activation of the delayed rectifier K(+) channel as well as the involvement of the Na(+)/Ca(2+) exchanger (NCX) and Na(+)/K(+) ATPase (ATPase). Roles of the K(+) channel, NCX and ATPase were determined using the specific blockers alpha-dendrotoxin, KB-R7943 and ouabain, respectively. Sodium azide (10mM) induced apoptosis in 40% of neurons. Inhibition of the AT(1) receptor with losartan (1 microM) facilitated angiotensin II mediated neuroprotection by reducing sodium azide-induced apoptosis 61.8 +/- 5.6%, while inhibition of the AT(2) receptor with PD123319 (1 microM) showed no neuroprotection. These results suggest that angiotensin II neuroprotection is mediated through the AT(2) receptor and requires inhibition of the AT(1) receptor in order to facilitate its effect. To determine the roles of delayed rectifier K(+) channel, NCX and ATPase cultures were pretreated with alpha-dendrotoxin (10nM), KB-R7943 (100 nM) and ouabain (100 nM), which significantly attenuated AT(2) receptor mediated neuroprotection. These findings further suggest that the mechanism of AT(2) receptor mediated neuroprotection is coupled to activation of the delayed rectifier K(+) channel, NCX and ATPase.

Animals↗

PubMed1992

Mutagenicity and antimutagenicity of hispidulin and hortensin, the flavonoids from Millingtonia hortensis L.

Studies of the mutagenicity and antimutagenicity of hispidulin and hortensin, the flavonoids from Millingtonia hortensis L. (Bignoniaceae), were performed using the liquid preincubation method of the Salmonella/microsome test. At the highest dose tested, 100 micrograms/plate, both compounds showed no mutagenicity and no cytotoxicity toward S. typhimurium strains TA98 and TA100 either in the presence or absence of S9 mix. However, these substances were antimutagens toward 2-aminoanthracene, aflatoxin B1 (in TA98), and dimethylnitrosamine (in TA100); but neither substance inhibited the direct mutagenic activity of 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide nor that of sodium azide in strains TA98 and TA100, respectively.

Aflatoxin B1↗

PubMed1985

Lithium ions inhibit function of low- but not high-affinity muscarinic receptors of murine neuroblastoma cells (clone N1E-115).

Murine neuroblastoma cells (clone N1E-115) possess both high- and low-affinity muscarinic receptors. The low-affinity muscarinic receptor, when stimulated, initiates the formation of cyclic GMP by activating the enzyme guanylate cyclase; whereas stimulation of the high-affinity receptor inhibits prostaglanding E1-mediated cyclic AMP formation by inhibiting the enzyme adenylate cyclase. We have reported that lithium ion (Li+) inhibits cyclic GMP formation mediated by the muscarinic receptor agonist, carbachol, in a concentration-dependent manner and that neither ammonium nor sodium ions have such an effect. We extended this study to show that Li+ was an apparently noncompetitive inhibitor of the low-affinity muscarinic receptor with an IC50(+/- SEM) = 13.6 +/- 0.8 mM. In addition, Li+ with a similar IC50 inhibited the cyclic GMP response in intact cells to sodium azide, which is thought to stimulate guanylate cyclase directly. Moreover, though Li+ was found to have a slight inhibitory effect on prostaglandin E1-stimulated cyclic AMP formation (15% inhibition at 10 mM), it had no effect on the function of the high-affinity muscarinic receptor in intact murine neuroblastoma cells.

Alprostadil↗

PubMed1986

Anthranilate-promoted iron uptake in Rhizobium leguminosarum.

Anthranilate promoted the uptake of ferric iron into iron-starved cells of Rhizobium leguminosarum GF160. The uptake system was a saturable function of the concentration of ferric anthranilate. It was characterized by an apparent Km of 6 microM and a Vmax of 1.6 nmol/min/mg cell protein. Uptake was temperature dependent and inhibited by the metabolic poisons arsenate and iodoacetate. The proton motive force may not be involved since no effect was demonstrated by the respiratory inhibitor sodium azide and by various uncouplers. The anthranilate-promoted iron uptake was lower for cells grown in increasing levels of available iron and the addition of anthranilic acid to low-iron cultures resulted in a stimulation of bacterial growth. During growth under iron deficiency, anthranilic acid was not assimilated by the cells.

Arsenates↗

PubMed1987

Ca2+-dependent ATP hydrolysis of the porcine intestinal brush-border membranes.

The brush-border membrane from the porcine small intestine possesses Ca2+-dependent ATPase activity. The Ca2+ stimulation of ATP hydrolysis by the membranes is biphasic with a high affinity (Km = 0.38 microM) and a low affinity (Km = 98.3 microM). Treatment of the membrane vesicles with n-heptylthioglucoside did not cause further increase of the Ca2+-ATPase activity. Mg2+ also stimulates the ATP hydrolysis in the absence of Ca2+ but decreases the Ca2+-ATPase activities at 0.59 and 200 microM free Ca2+. The Ca2+-ATPase activities are not inhibited by addition of vanadate, ouabain, sodium azide and alkaline phosphatase inhibitors (theophylline and L-phenylalanine), irrespective of the Ca2+ concentrations in medium. A specific calmodulin-inhibitor W-7 (up to 30 microM) also did not influence on the Ca2+-ATPase activities at 0.59 and 200 microM free Ca2+. The Ca2+-ATPase activities at 0.59 and 200 microM free Ca2+ show no specificity for ATP. ADP, GTP and CTP could also be used as substrates. From these results, it is suggested that the porcine intestinal brush-border membrane possesses Mg2+-independent Ca2+-ATPase activity and that the Ca2+-ATPase activities with biphasic responses for Ca2+ stimulation observed in the present study reside on the same protein. The physiological functions of the Ca2+-ATPase in the membranes, however, remain unknown at present.

Adenosine Triphosphate↗

PubMed1992

Proficiency of the Tradescantia-micronucleus image analysis system for scoring micronucleus frequencies and data analysis.

The Tradescantia-micronucleus (Trad-MCN) bioassay is an efficient short-term test for genotoxicity of pollutants. In order to increase the efficiency and to standardize the micronucleus (MCN) scoring process, an automated scoring system was developed using the principle of image analysis in computer science. This assemblage is called the Tradescantia-micronucleus image analysis (Trad-MCNIA) system. The MCN frequencies scored by this system were compared with those scored by human observation for its proficiency. A set of low MCN frequency (around 5 MCN/100 tetrads) slides prepared from a control group, a set of medium MCN frequency (around 20 MCN/100 tetrads) slides prepared from sodium azide treated plant cuttings and a set of high MCN frequency (around 50 MCN/100 tetrads) slides prepared from X-ray treated materials were used for this study. In the low MCN frequency slides, the Trad-MCNIA system scored about the same value as human observation. In the medium and high frequency slides, MCN frequencies scored by the system were lower than those scored by human observers. This discrepancy was corrected by increasing the power of the objective of the microscope in the system. The MCN frequencies scored by the system attained 90% congruity with those scored by human observers after the correction. The scoring speed of the system was about 3.5 times as fast as that by human observers, and the data could be statistically analyzed immediately after the data scores were recorded. Further improvements can be made by upgrading the video camera and the computer speed.

Azides↗

PubMed1983

Mobility and detergent extractability of acetylcholine receptors on cultured rat myotubes: a correlation.

On aneurally cultured rat primary myotubes, 10% of the acetylcholine receptors (AChR) are found aggregated and immobilized in endogenous clusters. The remaining receptors are diffusely distributed over the cell membrane and the majority of these are free to diffuse in the plane of the membrane. This study correlates the mobility of AChR (as measured with the fluorescence photobleaching recovery technique, FPR) with the detergent extractability of this receptor. Gentle detergent extraction of the cells removes the lipid membrane and the soluble cytoplasmic proteins but leaves an intact cytoskeletal framework on the substrate. Two studies indicate a correlation between mobility and extractability: (a) mobility of diffusely distributed AChR decreases as myotubes age in culture; previous work showed that extractability of AChR decreases as myotubes age in culture (Prives, J., C. Christian, S. Penman, and K. Olden, 1980, In Tissue Culture in Neurobiology, E. Giacobini, A. Vernadakis, and A. Shahar, editors, Raven Press, New York, 35-52); (b) mobility of clustered AChR increases when cells are treated with metabolic inhibitors such as sodium azide (NaN3); extractability of clustered AChR also increases with this treatment. From these results we suggest the involvement of a cytoskeletal framework in the immobilization of AChR on the cell surface.

Animals↗

PubMed1994

Cyclic AMP signalling pathway and trehalase activation in the fission yeast Schizosaccharomyces pombe.

The response of derepressed cells of Schizosaccharomyces pombe to the addition of glucose included a marked and reversible activation of neutral trehalase that was not produced in repressed cells. The protein synthesis inhibitor cycloheximide, the protonophore 2,4-dinitrophenol or the uncoupler sodium azide also enhanced trehalase activity in derepressed cells provided glucose was present in the incubation assays. However, only 2,4-dinitrophenol or cycloheximide was able to induce trehalase activation in repressed cells. Stimulation of trehalase by these compounds was preceded in all cases by a rapid increase in adenosine 3'-5'-cyclic monophosphate (cAMP) content. Since exogenous cAMP can activate trehalase both in repressed and derepressed growing cells, the results provide evidence for the existence of an induced cAMP signalling pathway in the fission yeast with several entries for trehalase activation. The correlation between cAMP increase and trehalase activation was not maintained when the enzyme was heat-shock-activated, supporting the concept that trehalase activity can be also enhanced in cells by another mechanism in which cAMP does not act as second messenger.

2,4-Dinitrophenol↗

PubMed1984

Panning separation for monoclonal antibody-specific T-cell subsets: effects of enzymes and metabolic inhibitors.

The mechanisms of "panning," a simple positive cell separation technique, were examined. Using monoclonal antibodies to "pan" for T cells (T101+, T4+, and T8+), we obtained enriched populations with 90% purity and viability from unfractionated human peripheral blood lymphocytes (PBL). We found that the "panning" method reflects an active process. It is sodium azide inhibitable and independent of the divalent cation concentration. Effective panning does not require capping, patch formation, or DNA synthesis. The cell yields are unaffected by mitomycin-C or microtubule and microfilament blocking reagents such as concanavalin A, colchicine, and cytochalasin B. This economical technique provides large numbers of functionally intact monoclonal antibody-specific cells within a relatively short time for further functional and biochemical characterization.

Adult↗