Absence Of Sodium Azide Research Articles

Aminopeptidase Modified Hydrolytic Enzymes to Improve the Efficiency of Sugar Production from Alkaline Pretreated Switchgrass

Cellulases and β-glucosidases (βGSD) are enzymes commonly used in the biofuel industry. In this study, smaller-sized variants were generated with aminopeptidase such that high catalytic capabilities were retained. Under the defined experimental conditions, the degree of hydrolysis was greater using cellulase substrates, compared to βGSD, based on ortho phthaldialdehyde (OPA) assay data (44% versus 15%). Proteolysis of cellulases was also evident based on sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) protein banding patterns seen after peptidase treatment. Residual cellulase activity was retained after peptidase hydrolysis (67% to 73%) based on standard filter paper assays. Peptidase treated cellulases and βGSD were then utilized for hydrolysis of alkaline-pretreated switchgrass (Panicum virgatum). Interestingly, the efficiency of the reaction, defined as milligrams of sugar produced per filter paper unit, was higher using truncated cellulases for bioprocessing reactions (~14%), especially in the absence of sodium azide. Conversely, incubation of βGSD with peptidase revealed minimal proteolysis with low impact on the efficiency of hydrolysis.

Modeling cytoplasmic release of encapsulated oligonucleotides from cationic liposomes

Transfection activity of antisense oligodeoxynucleotides (ODN)-loaded cationic liposomes is mainly restricted by uptake and ODN release into cytoplasm, which is difficult to evaluate in cell culture studies. Well-designed models of cellular membranes, aim of the present study, might facilitate investigation of such processes. In this investigation, a phosphorothioate ODN was actively encapsulated in a DODAP-containing cationic liposome by ethanol injection with 73% efficiency. ODN release was determined by fluorescence dequenching of FITC-ODN upon incubation of liposomes with early endosomal (EE), late endosomal (LE) and plasma membranes (PM) models. LE provided the highest release (up to 76%) in a temperature-dependent manner. Release by EE (<16%), total PM (<11%) and PM external layer (≈0) were not temperature sensitive. These differences are attributed to lipid charge, chain mobility, critical packing parameter and cholesterol content of the models. Intracellular distribution of FITC-ODN, determined by fluorescence microscopy and flowcytometry in the presence and absence of sodium azide, confirmed that liposomes were internalized mainly via endocytosis; hence inability of our PL models to simulate such active processes. Instead, release of ODN from endosomes into cytoplasm was pH-sensitive and in good agreement with model membrane studies in terms of amount and mechanism.

Involvement of adaptive mechanism and an energy-dependent efflux system of Pseudomonas sp. LE2 counteracting insecticide lindane

An adaptive mechanism and efflux-transport system of Pseudomonas sp. LE2 counteracting organochlorine insecticide lindane were investigated to understand the survival mechanism of this microorganism in lindane-contaminated environment. Changes in membrane fluidity and fatty acid composition of Pseudomonas sp. LE2 cells grown on glucose either in the presence or absence of lindane were determined. Considerably high ratio of total saturated to unsaturated fatty acids in cells grown on glucose in the presence of lindane was observed as compared to that in cells grown on glucose in the absence of lindane. Lindane significantly affected in vitro the membrane of Pseudomonas sp. LE2 cells by decreasing the fluorescence polarization of membrane probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) inserted into cytoplasmic membrane. Less fluidizing effect of lindane on the membranes of cells that had adapted to lindane was observed. Lindane transport was assessed by incubating cell suspensions with [ 14C]lindane either in the presence or absence of sodium azide, a membrane transport inhibitor, and by measuring the radioactivity in cell pellets. No time-dependent and steady-state increase in the radioactivity of [ 14C]lindane without sodium azide treatment was observed in cell pellets, suggesting simple diffusion mechanism for lindane accumulation in cells. Significant increase in the radioactivity was observed in cell pellets by sodium azide treatment. Sodium azide inhibited the efflux of lindane by Pseudomonas sp. LE2. These results suggest the view that Pseudomonas sp. LE2 capable of growing in the presence of lindane is able to adapt to the fluidizing action of lindane by incorporating the saturated fatty acid into membrane lipids, and eliminate lindane from cells by an energy-dependent efflux mechanism.

Ultraviolet irradiation of titanium dioxide in aqueous dispersion generates singlet oxygen

We previously reported that irradiation of titanium dioxide (TiO2) in ethanol generates both singlet oxygen (1O2) and superoxide anion (O2·-) as measured by EPR spectroscopy. The present study describes the production of reactive oxygen species upon irradiation of TiO2 in aqueous suspension as determined by EPR spectroscopy using 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TMP) and 5,5- dimethyl-pyrroline-N-oxide (DMPO). Photoproduction of 1O2 by suspended TiO2, detected as 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (4-oxo-TEMPO), was measured in water and deuterium oxide (D2O) in the presence or absence of sodium azide (NaN3) and under air or argon atmospheres. Production of a DMPO-OH adduct was examined in 4-oxo-TMP containing medium in the presence or absence of dimethyl sulfoxide (DMSO). The signal for the DMPO spin adduct of superoxide anion was not observed in aqueous conditions. Kinetic analysis revealed that 1O2 was produced at the surface of irradiated TiO2 in aqueous suspension as was observed in ethanol. Kinetic analysis revealed that the formation of DMPO-OH adduct reflects oxidation of DMPO by 1O2 rather than the trapping of the hydroxyl radical produced by the reaction of photo-exited TiO2 and water. The production of large amounts of 1O2 by TiO2 in aqueous suspension as compared to those in ethanol and possible formation of hydroxyl radical in aqueous suspension but not in alcohol, suggest that irradiation of TiO2 in aqueous environments is biologically more important than that in non-aqueous media.

Sorption coefficients of polycyclic aromatic hydrocarbons for two lake sediments: Influence of the bactericide sodium azide

AbstractSorption coefficients (Kps) of a series of polycyclic aromatic hydrocarbons (PAHs) were determined in two Dutch freshwater lake sediments to improve the insight into sorption in the aquatic environment. The influence of experimental factors, such as compound concentration, equilibration time, and influence of a bactericide on Kp, was studied. No significant influence of compound concentration on Kp was found. A contact time of 48 h was sufficient to reach equilibrium between PAHs in the sediment and water phase. In the absence of the bactericide, sodium azide, incomplete mass balances, and high Kp of low molecular weight PAHs were found that were not caused by volatilization from, photodegradation in, or sorption by the test system. In the presence of sodium azide, however, high mass balances and lower Kp were found for these compounds. This suggested that significant biodegradation of these compounds occurred in the water phase in the absence of sodium azide. The organic carbon‐normalized sorption coefficients (Koc) of the PAHs in Lake Oostvaardersplassen were two to three times higher than those in Lake Ketelmeer sediment. Although Koc of the PAHs differed for the two sediments, a clear relationship was found between Koc and the octanol/water partition coefficient for both sediments and between the Koc of both sediments. In conclusion, the methodology to determine Kp of biodegradable compounds should include the prevention of bacterial activity, e.g., by addition of sodium azide. Sorption of PAHs in the Dutch lake sediments is determined by the hydrophobicity of the PAH, the organic carbon content of the sediment, and one or more unspecified sediment‐specific characteristics.

Peroxidase-release associated with phagocytosis in Mytilus galloprovincialis haemocytes

Fluorescence spectra of the supernatant of adherent haemocyte monolayers from Mytilus galloprovincialis, supplemented with homovanillic acid or with a tyrosyl peptide glycylglycyltyrosine (GGY), were recorded before and after stimulation by zymosan. The formation of fluorescent derivatives was observed to have spectral characteristics similar to those of fluorescent compounds generated by the exposure of homovanillic acid or GGY to a horseradish peroxidase/hydrogen peroxide system in vitro. Lucigenine-enhanced chemiluminescence (CL luc) of M. galloprovincialis haemocytes stimulated by zymosan or by phorbol ester (PMA) was measured in the presence and absence of sodium azide, a peroxidase inhibitor. Sodium azide inhibited the CL luc of haemocytes stimulated by zymosan, an effective stimulus for myeloperoxidase secretion in human polymorphonuclear leukocytes, but not the CL luc of haemocytes stimulated by PMA, indicating the presence of peroxidases with some properties of myeloperoxidase, in adherent haemocytes from M. galloprovincialis.

Photoinduced transformation of wild-type and D96N-mutant 4-keto-bacteriorhodopsin gelatin films

Spectral and kinelic transformation studies of gelatin films based on 4-keto wild-type bacteriorhodopsin (BR) and 4-keto D96N mutant BR were carried out using absorbance spectroscopy. Spectral heterogeneity in the 400 nm range, assumed to be associated with the M-intermediate state, previously characterized for films of 4-keto wild-type BR, was observed in the 4-keto D96N mutant BR. This heterogeneity is associated to a greater extent with chromophore replacement than with amino acid replacement. The time constants of the M-decay are larger for all kinetic components for 4-keto D96N mutant in the absence of sodium azide. In addition, the contribution of the most long-lived component of the M-state decay is almost twice as large as for the film without sodium azide as it is for a film with sodium azide. This sodium azide effect on the M-state decay kinetics is opposite to that observed in the 4-keto wild-type BR. The comparison of the kinetics and spectral transformations of both pigments suggests that films containing 4-keto D96N mutant hold greater promise as media for information storage and retrieval.

Neutral endopeptidase of a human airway epithelial cell line recovers after hypochlorous acid exposure: dexamethasone accelerates this by stimulating neutral endopeptidase mRNA synthesis.

Hypocholorous acid (HOCl) exposure of Calu-1 cells in situ leads to a relatively rapid and substantial decrease in whole cell neutral endopeptidase (NEP) activity that may result from the internalization of NEP from plasma membrane surfaces. To confirm this, and to assess the time course of changes in cell NEP after oxidant exposure and the potential influence of corticosteroid treatment on these, we evaluated Calu-1 NEP activity by high performance liquid chromatography and NEP-specific mRNA over the ensuing 48 h after HOCl in the presence or absence of 1 microM dexamethasone. Cells, grown to confluency in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, were exposed for 5 min to 100 microM HOCl and then maintained in culture for 48 h thereafter. Before exposure, some cell plates were cooled to 4 degrees C and/or incubated for 5 min in 1 mM sodium azide. In some experiments, post-HOCl changes in NEP-specific mRNA in the presence or absence of dexamethasone were also evaluated using Northern blot analysis. We found that sodium azide at 4 degrees C totally blocked the effect of HOCl on Calu-1 NEP (n = 6). In the absence of sodium azide, NEP activity spontaneously recovered to preexposure levels within 24 h. This recovery occurred 6 h earlier in the presence of 1 microM dexamethasone. Furthermore, dexamethasone increased NEP activity at 24 and 48 h after HOCl. Northern blot analysis indicated that NEP-specific mRNA did not change during spontaneous recovery, but was increased by dexamethasone 24 h after HOCl.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-dependent Ca 2+ accumulation in intracellular membranes of guinea pig macrophages after saponin treatment

Ca 2+ transport system in the intracellular membranes was studied by using saponin-treated macrophages of the guinea pig, in which the plasma membranes could be selectively destroyed. Saponin-treated macrophages could accumulate 3.1 nmoles Ca 2+ 4 × 10 6 macrophages in the presence of Mg-ATP and sodium azide with an apparent affinity constant of 6 × 10 6 M −1. In the absence of sodium azide, the value of Ca 2+ uptake of saponin-treated macrophages was 95 nmoles/4 × 10 6 macrophages, and its affinity constant for Ca 2+ was 3 × 10 5 M −1. Saponin-treated macrophages may be suitable for the studies of Ca 2+ transport systems in the intracellular membranes.

Activation of rat liver guanylate cyclase by proteolysis.

Guanylate cyclase activity (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.), measured in purified rat liver plasma membranes, was markedly increased by treatment with various purified proteases. The effect was maximal with trypsin, alpha-chymotrypsin, papain, and thermolysin (6- to 8-fold increase with 5 to 20 microgram of protease/ml) and lower with subtilisin and elastase (3- to 4-fold increase). The activation was due to an increase in the maximal velocity of the cyclizing reaction. No modification was observed either in the apparent affinity for the substrate MnGTP or in the cooperative behavior of the enzyme kinetics which displayed Hill coefficients of 1.6 for both basal and activated states. The Triton X-100-dispersed guanylate cyclase remained sensitive to papain, which suggests that the action of proteases was not restricted to an indirect action upon the membranous environment of the guanylate cyclase. In contrast, the cytosolic soluble guanylate cyclase, assayed in the presence or absence of sodium azide, was absolutely insensitive to papain. Thus, proteolysis represents a previously undescribed mechanism for activating membranous guanylate cyclase systems, which might be of importance in the physiological regulation of this enzyme.

Fatty Acid beta-Oxidation System in Microbodies of n-Alkane-Grown Candida tropicalis

Localization of fatty acid beta-oxidation system in microbodies of Candida tropicalis cells growing on n-alkanes was studied. Microbodies isolated from the yeast cells showed palmitate-dependent activities of NAD reduction, acetyl-CoA formation and oxygen consumption. When sodium azide, an inhibitor of catalase, was added to the system, palmitate-dependent formation of hydrogen peroxide was observed. Stoichiometric study revealed that two moles of NAD were reduced per one mole of oxygen consumed in the absence of sodium azide and the presence of the inhibitor doubled the oxygen consumption by microbodies without an appreciable change in NAD reduction. These results indicate that the yeast microbodies contain beta-oxidation system of fatty acid, and that catalase located in the organelles participates in the degradation of hydrogen peroxide to be formed at the step of dehydrogenation of acyl-CoA.

Effects of Azide on Photophosphorylation in Isolated Spinach Chloroplasts

AbstractThe influence of sodium azide on open‐chain and flavine mononucleotide mediated cyclic photophosphorylation in isolated spinach chloroplasts was investigated under anaerobic conditions. Open chain phosphorylation was completely inhibited with DCMU both in the presence and absence of sodium azide in the experimental medium. Flavine mononucleotide mediated photophosphorylation was only slightly inhibited by DCMU in the absence of sodium azide but inhibited in two steps by increasing amounts of DCMU when sodium azide was present in the medium. The first step can be explained as being mainly an effect of DCMU on an open chain electron transport, with water and H2O2 as electron donors and with flavine mononucleotide — kept in an oxidized state by sodium azide — as the electron acceptor. The second step, as well as the comparatively insensitivity to DCMU in the absence of sodium azide, depends on cyclic photophosphorylation mediated by flavine mononucleotide.

Effects of mitogens on sodium-potassium transport, 3H-ouabain binding, and adenosine triphosphatase activity in lymphocytes

The effect of various mitogens was studied on sodium (Na +) potassium (K +) transport, 3H-ouabain binding, and adenosine triphosphatase (ATPase) activity in human and sheep peripheral lymphocytes. Concanavalin A (ConA), phytohemagglutinin (PHA), horse anti-lymphocyte serum (ALS), and anti-IgG antisera, in order of decreasing potency, stimulated in particular the ouabain-sensitive K + pump influx, while the cardiac glycoside-insensitive K + leak flux was only slightly affected. Sheep lymphocytes primed in vivo with human IgG as antigen also responded with K + pump flux activation when exposed to the antigen in vitro. Both PHA and ConA also stimulated active Na + efflux in human lymphocytes. Apparently these mitogens activate the Na +K + pump system in the lymphocyte membrane—an assumption supported by the finding of a significant activation of the ouabain-sensitive Na +K +-ATPase. From rate studies of 3H-ouabain binding carried out at 37 °C in presence and absence of sodium azide, and at 0 °C, it is concluded that PHA alters the rate of ouabain uptake to these cells. Thus PHA may alter the affinity of the pump for ouabain, equivalent to an increased cation turnover per pump site. However, our findings do not completely discount the possibility that PHA also increases the total number of ouabain molecules bound and therefore of Na +K + pumps.