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PubMed1973

Abolition of swarming of Proteus by p-nitrophenyl glycerin: application to blood agar media.

Comparative plate counts were made of Staphylococcus aureus and Streptococcus pyogenes growing on blood agar supplemented with individual chemicals to abolish the swarming of Proteus. B-phenylethanol, sodium azide, and p-nitrophenyl glycerin (PNPG) were used as anti-swarm agents. Each anti-swarm agent effectively abolished swarming for 24 h, but azide failed to control swarming for longer periods of incubation. In addition, azide displayed growth inhibition towards the staphylococci and streptococci resulting in no hemolysis and reduced viable cell numbers with the streptococci. Phenylethanol showed reduced viable cell numbers with the streptococci and unreliable hemolytic reactions. At 0.1 to 0.3 mM, PNPG proved to be a superior anti-swarm agent in that it showed no growth inhibition and allowed normal hemolysis, but abolished swarming for extended periods of time. When laboratory strains of Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa. Listeria monocytogenes, and Vibrio cholerae were screened on a blood agar medium containing 0.1 mm PNPG, they displayed similar growth and hemolytic characteristics to the identical medium without PNPG.

Agar↗
PubMed2001

Expression of the reduced folate carrier SLC19A1 in IEC-6 cells results in two distinct transport activities.

Intestinal absorption of folates has been characterized as a facilitative process with a low pH optimum. Studies with intestinal epithelial cells have suggested that this activity is mediated by the reduced folate carrier (RFC1). In this paper, we report on folate transport characteristics in an immortalized rat IEC-6 cell line that was found to exhibit the predominant influx activity for methotrexate (MTX) at pH 5.5 with a low level of activity at pH 7.4. Transfection of this cell line with an RFC1 construct resulted in clones exhibiting increased MTX uptake at both the pHs and high folic acid uptake only at the low pH. For the two clones with the highest level of transport activity, relative MTX influx at the two pHs was reversed. Moreover, the low pH MTX influx activity ([MTX](e) = 0.5 microM) was markedly inhibited by 20 microM folic acid while influx at neutral pH was not. Furthermore, in the presence and absence of glucose at low pH, MTX and folic acid influx activity was inhibited by azide, while MTX influx at pH 7.4 was stimulated by azide in the absence of glucose but was unchanged in the presence of glucose and azide. This was contrasted with the results of transfection of the same RFC1 construct into an L1210 murine leukemia cell line bearing a nonfunctional endogenous carrier. In this case, the activity expressed was only at pH 7.4. These data indicate that RFC1 can exhibit two distinct types of folate transport activities in intestinal cells that must depend on tissue-specific modulators.

Animals↗
PubMed1984

Histochemical localization of NADP-dependent dehydrogenase activity with four different tetrazolium salts.

The properties of the four most commonly used tetrazolium salts, neotetrazolium, nitro blue tetrazolium (nitro-BT), tetranitro-BT, and 2-(2-benzothiazolyl-3-(4-phthalhydrazidyl)-5-styryl-te trazolium (BPST), have been compared for their effects on the localization of nicotinamide adenine dinucleotide phosphate (NADP)-dependent dehydrogenases under optimal incubation conditions in cryostat sections of rat liver. Glucose-6-phosphate dehydrogenase has been selected as an example of these dehydrogenases. It was found that the use of nitro-BT and tetranitro-BT, unlike neotetrazolium and BPST, in combination with an exogeneous electron carrier and azide, resulted in localization patterns in agreement with the sites of activity as determined by microchemical techniques. In the absence of an intermediate carrier the localization was very similar to that of NADPH cytochrome c (P450) reductase as demonstrated immunocytochemically. BPST did not properly localize dehydrogenase activity, most probably because of the redistribution of formazan, due to its lack of firm substantivity. Neotetrazolium reduction in nitrogen gave the localization pattern, both in the presence and absence of carrier, of the reductase, suggesting that the transfer of reducing equivalents from the exogenous electron carrier to neotetrazolium proceeds via cellular electron transport systems. The reduction of nitro-BT and tetranitro-BT via intermediate carriers was oxygen sensitive in parenchymal cells, but not in the non-parenchymal liver cells. This oxygen sensitivity could be blocked by azide. With neotetrazolium, oxygen inhibited both carrier-mediated and carrier-independent reactions, effects that were not reversed with azide. Possible mechanisms of action between oxygen, reduced carriers, and tetrazolium salts are discussed.

Animals↗
PubMed1992

Adenosine triphosphate-sensitive K+ channels may not be the sole regulators of glucose-induced electrical activity in pancreatic B-cells.

Stimulation of insulin release by glucose requires Ca2+ influx in pancreatic B-cells. This influx occurs during phases of electrical activity (slow waves of membrane potential with superimposed spikes) that can be monitored with intracellular microelectrodes. It has been suggested that closure of ATP-sensitive K+ channels contributes to the increase in electrical activity (and, hence, in Ca2+ influx and insulin release) produced by suprathreshold (greater than 7 mM) concentrations of glucose. If this is the sole mechanism of control, the decrease in electrical activity that follows a decrease in glucose concentration should be mimicked by opening these ATP-sensitive K+ channels. This was achieved by diazoxide, which selectively and directly acts at the channel level (without decreasing B-cell metabolism), and azide, which indirectly opens the channels by inhibiting mitochondrial ATP production. Stepwise lowering of the glucose concentration from 15 to 8 mM progressively decreased electrical activity in B-cells. This decrease was characterized by a shortening of the slow waves and a lengthening of the intervals between the slow waves, with little change in slow wave frequency. Similar changes followed the addition of azide (250-750 microM) to a medium containing 15 mM glucose. In contrast, in the presence of 15 mM glucose, diazoxide (5-20 microM) considerably increased the interval duration, but did not shorten the slow waves, thus causing a marked fall in slow wave frequency. In B-cells persistently depolarized by 30 mM glucose, diazoxide restored slow waves and intervals that were much longer than those recorded when the same cells were stimulated by 15 mM glucose. In conclusion, decreasing mitochondrial ATP production with azide is more able to reproduce the effects of a decrease in glucose concentration on electrical activity in B-cells than a direct pharmacological opening of ATP-sensitive K+ channels with diazoxide. This suggests that ionic channels other than ATP-sensitive K+ channels are under metabolic control and may contribute to the regulation of electrical activity by glucose.

Adenosine Triphosphate↗
PubMed2000

Oxygen uptake photosensitized by disorganized chlorophyll in model systems and thylakoids of greening barley.

Light-dependent oxygen uptake was observed and studied in thylakoids from early greening barley in comparison to oxygen uptake in chlorophyll solutions and in thylakoids from fully green leaves. Substantial oxygen uptake was observed in chlorophyll solutions supplemented with tryptophan, histidine, ascorbic acid or linoleic acid. This uptake was diminished by adding azide, beta-carotene and alpha-tocopherol, which are specific singlet-oxygen quenchers. Illuminated thylakoids from greening barley also exhibited marked oxygen uptake that, likewise, was strongly quenched by azide. In comparison, azide was found not to affect oxygen uptake that is associated with the methyl viologen-catalyzed Mehler reaction. It is reasoned that in the first two cases the oxygen uptake arises from chlorophyll-photosensitized activation of oxygen to the singlet state and its consumption by exogenous or endogenous substrates. In greening, we propose that disorganized chlorophyll photo-sensitizes the oxygen uptake.

Chlorophyll↗
PubMed2002

Illumination accelerates the decay of the O-intermediate of pharaonis phoborhodopsin (sensory rhodopsin II).

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II [psRII]) is a member of the archaeal rhodopsin family and acts as a repellent phototaxis receptor of Natronobacterium pharaonis. Upon illumination, ppR is excited and undergoes a linear cyclic photoreaction, namely, a photocycle that constitutes photointermediates such as M- and O-intermediates (ppRM and ppRO, respectively). Under a constant background illumination (>600 nm) that irradiates ppRO, the decay rate of the flash-induced ppRO increased with an increase in the background light intensity, indicating the photoreactivity of ppRO. Azide did not influence the light-accelerated ppRO decay, but the time required for the cycle to be completed became shortened in an azide concentration-dependent manner because of acceleration of ppRM decay. Hence, the turnover rate of photocycling increased appreciably in the presence of both the background illumination and the azide. The observation reported previously (Schmies, G. et al. 2000, Biophys. J. 78:967-976) is discussed in connection with the present observations.

Archaeal Proteins↗
PubMed2000

An antiprion effect of the anticytoskeletal drug latrunculin A in yeast.

Prions are infectious aggregation-prone isoforms of the normal proteins, supposedly able to seed aggregation of the normal cellular counterparts. In vitro, prion proteins form amyloid fibers, resembling cytoskeletal structures. Yeast prion [PSI], which is a cytoplasmically inherited aggregated isoform of the translation termination factor Sup35p (eRF3), serves as a useful model for studying mechanisms of prion diseases and other amyloidoses. The previously described interaction between Sup35p and cytoskeletal assembly protein Sla1p points to the possible relationships between prions and cytoskeletal networks. Although the Sup35PSI+ aggregates do not colocalize with actin patches, we have shown that yeast cells are efficiently cured of the [PSI] prion by prolonged incubation with latrunculin A, a drug disrupting the actin cytoskeleton. On the other hand, treatments with sodium azide or cycloheximide, agents blocking yeast protein synthesis and cell proliferation but not disrupting the cytoskeleton, do not cause a significant loss of [PSI]. Moreover, simultaneous treatment with sodium azide or cycloheximide blocks [PSI] curing by latrunculin A, indicating that prion loss in the presence of latrunculin A requires a continuation of protein synthesis during cytoskeleton disruption. The sodium azide treatment also decreases the toxic effect of latrunculin A. Latrunculin A influences neither the levels of total cellular Sup35p nor the levels of chaperone proteins, such as Hsp104 and Hsp70, which were previously shown to affect [PSI]. This makes an indirect effect of latrunculin A on [PSI] via induction of Hsps unlikely. Fluorescence microscopy detects changes in the structure and/or localization of the Sup35PSI+ aggregates in latrunculin A-treated cells. We conclude that the stable maintenance of the [PSI] prion aggregates in the protein-synthesizing yeast cells partly depends on an intact actin cytoskeleton, suggesting that anticytoskeletal treatments could be used to counteract some aggregation-related disorders.

Actins↗
PubMed1991

Influence of extracellular pH on the accumulation and cytotoxicity of N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea in human cell lines.

The effect of extracellular pH (pH(e)) on the accumulation and cytotoxicity of the diarylsulfonylurea antitumor agent N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (MPCU) has been examined. In a human colon adenocarcinoma cell line, GC3/C1, the initial rate of uptake of [3H]MPCU (2.4 microM) was increased by 4.5-fold as pH(e) was reduced from 7.4 to 6.5. Steady state levels of MPCU were inversely proportional to pH(e) and were 5-fold greater at pH 6.0 compared to 7.4. Similar results were obtained using Rh30 cells derived from an alveolar rhabdomyosarcoma. MPCU rapidly re-equilibrated after achieving steady state when pH(e) was altered, indicating that MPCU was not tightly bound within cells. In both cell lines, the uncoupling agent, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), significantly reduced (GC3/C1) or completely inhibited (Rh30) accumulation of MPCU at each pH(e) examined. Sodium azide had the same effect on the accumulation of MPCU as FCCP. The effects of FCCP and azide appeared to be due to collapse of the pH differential across the mitochondrial inner membrane rather than the gradient across the plasma membrane. As extracellular pH (pH(e)) decreased, intracellular pH(pH(i)) also decreased in GC3/C1 cells, such that the greatest pH differential (pH(i) - pH(e)) was 0.2 units at pH(e) 6.0. Neither FCCP nor azide significantly altered this pH gradient, indicating a minor role, if any, for the plasma membrane pH gradient in accumulation of MPCU in GC3/C1 cells. The effect of pH(e) (7.4 to 6.0) on cytotoxicity of MPCU was determined after exposure of cells for 4 hr to various concentrations of MPCU in the presence of 10% fetal bovine serum. Decreasing the pH(e) from 7.4 to 6.0 increased the potency of MPCU by 4.7- and 4.5-fold in Rh30 and GC3/C1 cells, respectively. In cells exposed to drug/pH(e) combinations that resulted in 50% reduction in colony forming potential, the steady state levels of [3H]MPCU were similar (range 8.8 +/- 0.9 to 10.56 +/- 0.6 nmol/10(6) cells). These results demonstrate that decrease of pH(e) significantly enhanced the uptake of MPCU accumulation into an FCCP/azide-sensitive compartment, and cytotoxicity of this agent. These data further support the hypothesis that sequestration of diarylsulfonylureas into the FCCP/azide-sensitive compartment (probably mitochondria) was associated with its cytotoxicity. The role of pH(e) in determining therapeutic selectivity of diarylsulfonylureas is discussed.

Adenocarcinoma↗
PubMed1980

The aminoglycoside antibiotics.IV. Synthesis of aminodeoxy analogs of neamine.

An efficient synthesis of the key 3',4'-galacto epoxide intermediate 4 obtained from the known 5,6-O,O'-cyclohexylidene-N,N'-bis-(methoxycarbonyl)-4-O-[2,6-bis(methoxycarbony lamino)-alpha-D-glucopyranosyl]-2-deoxystreptamine (5) is described. Treatment of this epoxide with sodium azide, followed by reduction and acetylation, yielded the protected4'-amino-4'-deoxyneamine 18 (3',4'-diequatorial), whereas treatment with ammonia followed by acetylation yielded the protected 3'-amino-3'-deoxyneamine analog 19 with a diaxial configuration of its 3' and 4' positions. Reaction of the previously described protected 3',4'-allo epoxide 3 with sodium azide yielded separable mixtures of the protected 3'-amino-3'-deoxyneamine 14 and the protected diaxial 4'-amino-4'-deoxyneamine isomer 13, the ratios of products depending on the solvent temperature. Structural assignments for 13, 14, 18 and 19 were based on their PMR spectra. An additional 4'-amino-4'-deoxyneamine analog (24) with an axial configuration as its 4' position was also prepared by azide displacement of an approximately protected 4'-methanesulfonyl neamine intermediate 10. The five protected isomers were deblocked to yield a series of aminodeoxyneamine analogs (15, 16, 20, 21 and 25), all of which were less active in vitro than neamine against a group of Gram-positive and Gram-negative bacteria.

Bacteria↗
PubMed1992

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.

Circular Dichroism↗
PubMedDate not supplied

[Identification and catalytic properties of Mg2+-dependent ATP-hydrolase of cytoplasmic membrane of Bacillus sp. B4253 capable of gold accumulation].

Ca2+-independent, Mg2+-dependent ATP-hydrolase fermentative activity consisting of two components--azide-sensitive and azide-resistant ones has been identified in cytoplasmic membrane of Bacillus sp. B4253 capable to gold accumulation in ionic and colloid forms. The authors have characterized properties of the azide-resistant component of ATP-hydrolase reaction: dynamics of accumulation of one of the reaction products--inorganic phosphate P(i); dependence of ATP hydrolysis rate on the membrane protein content; pH-dependence; sensitivity of ATP-hydrolase activity to the change of reagents (ATP, Mg2+) concentration, as well as to the effect of some specific and nonspecific inhibitors of ion-transporting Mg2+-dependent ATP-hydrolase systems (ouabain, tapsigargin, eocine Y, La ions). It is supposed that the obtained experimental data can be used for the following study of molecular and membrane mechanisms of gold accumulation in Bacillus sp. B4253.

Adenosine Triphosphate↗
PubMed1991

Characterization of alternative respiratory pathways in the yeast Schwanniomyces castellii by the study of mutants deficient in cytochromes a+a3 and/or b.

After a general review of the proposed mechanisms and physiological roles of the alternative respiratory pathways found in various organisms, the studies are focussed on the amylolytic yeast Schwaniomyces castellii. In addition to the cytochrome chain, the wild type presents two alternative pathways insensitive to antimycin A. One is salicylhydroxamic acid (SHAM)-sensitive and azide-insensitive; the other is SHAM-insensitive and sensitive to high azide concentration. Conditions for mutagenesis and screening are described, which allow isolation of mutants deficient in cytochromes a+a3 and/or b in this yeast previously classified as petite negative. The relative proportions of the alternative respiratory pathways are compared in the wild type and mutant strains following inhibition by SHAM and azide at optimal concentration as determined by iso-inhibition curves. The growth of the cytochrome deficient mutants on citrate, a non-fermentable carbon source, and the ability of the wild type to grow on citrate+antimycin A, after a lag of about 10 h, indicate an involvement of the alternative pathway(s) in energy production. Rotenone sensitivity of respiration and ATP level confirm the presence of a functional phosphorylation site 1. The role of each alternative respiratory pathway in energy production is discussed.

Cyanides↗
PubMed1991

Mutations in Ser174 and the glycine-rich sequence (Gly149, Gly150, and Thr156) in the beta subunit of Escherichia coli H(+)-ATPase.

A sequence motif in the beta subunit of Escherichia coli F1 (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residue 149-156, where conserved residues are underlined) is one of the glycine-rich sequences found in many nucleotide binding proteins. In this study, we constructed a plasmid carrying all the F0F1 genes. This plasmid gave the highest membrane ATPase activity so far reported. Substitution of beta Gly149 by Ser suppressed the effect of the beta Ser174----Phe mutation (defective H(+)-ATPase), but beta Gly150----Ser substitution did not have this effect. A single mutation (beta Gly149----Ser or beta Gly150----Ser) gave active enzyme with altered divalent cation dependency and azide sensitivity: the beta Gly149----Ser mutant enzyme had 100-fold lower azide sensitivity and essentially no Ca(2+)-dependent activity, but had the wild-type level of Mg(2+)-dependent activity with active oxidative phosphorylation. Introduction of a beta Gly149----Ser or beta Gly150----Ser mutation with the beta Ser174----Phe mutation also lowered the Ca(2+)-dependent activity and azide sensitivity. Consistent with our previous findings (Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 21279-21284), a beta Thr156----Ala or Cys mutation impaired ATPase activity, suggesting that the hydroxyl moiety at position 156 is essential for the catalytic activity. The possible location of the catalytic site including divalent cation binding site(s) is discussed.

Amino Acid Sequence↗
PubMed1987

Immunosuppression by activated human neutrophils. Dependence on the myeloperoxidase system.

An in vitro model system was used to define the mechanism of interaction between human neutrophils and lymphocytes. Blood mononuclear leukocytes were exposed to purified neutrophils in the presence of a neutrophil-activating agent (phorbol ester, lectin, or opsonized particle). The treated mononuclear cells displayed a marked decrease in both natural killer activity and mitogen-dependent DNA synthesis, but no change in viability. This functional suppression was dependent on neutrophil number, stimulus concentration, and duration of exposure. Lymphocytes were protected by addition of catalase, but not superoxide dismutase. Neutrophils defective in oxidative metabolism (chronic granulomatous disease) failed to suppress lymphocyte function unless an H2O2-generating system, glucose oxidase plus glucose, was added. The patients' neutrophils provided a factor, possibly myeloperoxidase, which interacted with the glucose oxidase system. The immunosuppressive effect of normal neutrophils was diminished when chloride was omitted from the cultures and was enhanced when chloride was replaced by iodide. Myeloperoxidase-deficient neutrophils were partially defective in suppressing lymphocytes and this was corrected by addition of purified myeloperoxidase. Paradoxically, azide caused enhancement of suppression that depended on the neutrophil oxidative burst, but not on myeloperoxidase and was mediated at least in part by an effect of azide on the target mononuclear leukocytes. These data indicate that suppression of lymphocyte function by activated neutrophils is mediated by the secretion of myeloperoxidase and H2O2 that react with halides to form immunosuppressive products. Moreover, the mononuclear leukocytes contain an azide-sensitive factor, probably catalase, which provides partial protection against injury by neutrophil products. These dynamic interactions may be important local determinants of the immune response.

Cell Survival↗
PubMed1987

The role of reactive oxygen intermediates in nonspecific monocyte cytotoxicity induced by immune complexes.

Normal human monocytes were induced to lyse nonsensitized target cells when triggered by precipitating immune complexes (IC) or soluble heat-aggregated IgG (HAIgG). Catalase, azide, cyanide and three aminoacids employed as quenchers of ClO, significantly inhibited this nonspecific cytotoxicity (NSC), suggesting an important role for the myeloperoxidase (MPO) system. However, HO and/or 1O2 may also be involved in the lysis, since certain scavengers of these species such as mannitol, benzoate, ethanol and histidine, as well as superoxide dismutase (SOD), partially inhibited NSC. Moreover, cyanide and azide were unable to completely abrogate this lytic activity. When NSC was compared to antibody dependent cellular cytotoxicity (ADCC), it was found that neither catalase nor oxygen-species scavengers affected ADCC while azide and cyanide significantly enhanced it. Antibody-coated target cells were also destroyed by IC-triggered monocytes. However, kinetic analysis and studies on the capacity of catalase to inhibit the lysis demonstrated that it was mediated through a NSC-like mechanism. The cytotoxic system described in this report offers a suitable model to study in vitro alternative lytic mechanisms triggered through monocyte receptors for the Fc portion of IgG (Fc gamma R).

Antibody-Dependent Cell Cytotoxicity↗
PubMedDate not supplied

[Alcohol oxidation by Candida guilliermondii yeasts grown on hexadecanol].

A number of enzyme systems involved in the first steps of hexadecane oxidation can be induced by hexadecanol, an intermediate product of hexadecane degradation. It has also been found that, in Candida guilliermondii cells and in their mitochondrial fraction, an oxidase system is induced when the cells are grown on hexadecanol. This system is similar to that in cells grown on hexadecane; it oxidises higher alcohols at a high rate and is not inhibited by the inhibitors of the man phosphorylating respiration chain. The membrane-bound alcohol dehydrogenase and aldehyde dehydrogenase activities resistant to pyrazole, an inhibitor of cytosol ethanol dehydrogenase, are induced together with the oxidase activity when the cells are grown on hexadecanol as well as on hexadecane. The oxidation of higher alcohols by whole cells is entirely inhibited by azide although their oxidation by mitochondria is resistant to the action of azide; apparently, azide inhibits the transport of alcohols into the cell.

Alcohol Oxidoreductases↗
PubMed1982

Arming of mononuclear phagocytes by eosinophil peroxidase bound to Staphylococcus aureus.

When eosinophil peroxidase (EPO) was incubated with Staphylococcus aureus (staph) approximately 40% remained firmly bound to the bacteria as indicated by guaiacol assay. The staph-EPO complex was killed readily by concentrations of H2O2 and a halide that did not alter the viability of control (non-EPO-coated) organisms or organisms pretreated with another basic protein, protamine. This toxic effect was abolished by omitting either H2O2 or halide and was inhibited by azide, cyanide, or aminotriazole. Peroxidase activity was evident on the surface of the organism after ingestion by rabbit blood monocytes. The staph-EPO complex was killed at a greater rate by both rabbit blood monocytes and alveolar macrophages than were control organisms, an effect that did not appear to be secondary to improved phagocytosis. The peroxidase inhibitor azide inhibited the killing of staph-EPO by these mononuclear phagocytes without affecting the killing of control staphylococci; in the presence azide, the killing curve with staph-EPO returned to that seen with control organisms. Further, aerosolized EPO-coated organisms were cleared more rapidly from rat lung than were uncoated organisms or organisms pretreated with protamine. These findings suggest mononuclear phagocytes can utilize EPO bound to the surface of ingested microorganisms to enhance their microbicidal activity.

Animals↗
PubMed1994

A simple in vitro method to detect singlet oxygen and to compare photodynamic activity using alkaline phosphatase.

A simple, sensitive and reliable in vitro method based on photodynamic inactivation of alkaline phosphatase to detect singlet oxygen and for evaluating relative photosensitizing efficiencies of photosensitizers such as hematoporphyrin (Hp) and phthalocyanines has been developed and compared with photobleaching of p-nitroso dimethyl aniline (RNO) and photooxidation of L-tryptophan. Inactivation of alkaline phosphatase is dependent both on light fluence and sensitizer concentration. Scavengers like mannitol and azide anion indicated the involvement of singlet oxygen in the deactivation of alkaline phosphatase, since azide anion provided concentration dependent protection whereas mannitol had no effect and that compared to ordinary water, photoinactivation of alkaline phosphatase was three times higher in 65% D2O. Alkaline phosphatase appears to be resistant to free radical attack (particularly to OH radicals) since hydrogen peroxide alone or in presence of ferrous ions did not reduce the enzyme activity and mannitol or azide anion gave no significant protection when alkaline phosphatase was irradiated with Co-60 gamma rays up to 2 K Gy. With the present method using red light, the chloroaluminium phthalocyanine sulphonates prepared by sulphonation showed higher and the corresponding condensation product lower photodynamic activity; Hp being intermediate and Mn- and Gd-phthalocyanines had no photodynamic activity.

Alkaline Phosphatase↗