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PubMed1979

The influence of some metabolic inhibitors on phagocytic activity of mouse macrophages in vitro.

The action of different metabolic inhibitors on phagocytosis by macrophages from mouse peritoneal exudate cultured in vitro was studied. The following metabolic inhibitors were tested: sodium iodoacetate, sodium fluoride, sodium fluoroacetate, sodium malonate, 2-4-dinitrophenol, sodium azide, ouabain and cycloheximide, all at the concentration of 10(-3) M. Iodoacetate caused a strong inhibitory effect on phagocytosis; this observation confirms that glycolysis is the main source of energy for the phagocytic process. On the contrary, fluoride, although it is an effective inhibitor of glycolysis, did not exert any effect. This difference may be explained by the fact that sodium fluoride blocks anaerobic glycolysis only in vitro at an unphysiological temperature (0 degrees C). Fluoroacetate and malonate, two compounds which interfere with the Krebs cycle, did not inhibit phagocytosis, but it is known that the Krebs cycle activity is poorly developed in the macrophagic cells. Sodium azide and 2-4-dinitrophenol, two inhibitors of oxidative phosphorylation, showed an effect on phagocytosis only after 3 h of contact with the cell cultures. Ouabain blocks Na+ and K+ transport across the plasma membrane and, probably, it inhibited phagocytosis by interfering with the movements of the cell membrane. Finally, the mode of action of cycloheximide on phagocytosis is uncertain. This compound inhibits the protein synthesis and, perhaps, it can act by preventing the renewal of the cell membrane.

Animals↗

PubMedDate not supplied

Fast and sensitive chemiluminescence determination of H2O2 concentration in stimulated human neutrophils.

A fast and sensitive chemiluminescence assay for the determination of H2O2 in stimulated neutrophils without the use of enzymes was developed. The method is based on the oxidation of luminol by hypochlorous acid. The chemiluminescence of this reaction is highly dependent on the concentration of hydrogen peroxide. Changes in H2O2 concentration in PMA-stimulated neutrophils were followed by injection of NaOCI to cell suspension at different times after cell stimulation. The short integration time of 2 s permits calculation of actual concentrations of H2O2 without influence of H2O2 decomposition by cellular enzymes or newly produced H2O2 due to dismutation of superoxide anion radicals. Concentrations of H2O2 were diminished by catalase and enhanced by sodium azide owing to inhibition of cellular catalase and myeloperoxidase. Changes in H2O2 concentration upon stimulation could be observed at 3000 cells/mL.

Azides↗

PubMed1999

Effects of low oxygen concentrations and metabolic inhibitors on proteoglycan and protein synthesis rates in the intervertebral disc.

The intervertebral disc is the largest asvacular structure in the body; consequently, there are steep gradients in O2 concentration, with PO2 falling to as low as 1% O2 in the centre of the disc. We investigated the effect of O2 concentration on the rates of O2 consumption, lactate production, and sulphate and proline incorporation in bovine caudal discs. We also investigated the effects of metabolic inhibitors of energy production pathways on tracer incorporation. Samples from the outer annulus and nucleus pulposus were incubated for 24 hours in 1-21% O2. Rates were measured during the last 4 hours of incubation. As O2 concentration was reduced from 10 to 1% O2, O2 consumption rates fell by around 75% and lactate production rates almost doubled; the bovine discs thus showed a positive progressive Pasteur effect. Incorporation rates of [3H]proline and [35S]sulphate were lowest at 1% O2. In the nucleus, but not in the outer annulus, the rate of [35S]incorporation peaked at 5% O2, where it was 30% greater than at 21% O2 and 150% greater than at 1% O2. The competitive glycolysis inhibitor 2-deoxyglucose, the oxidative phosphorylation uncoupler 2,4-dinitrophenol, and the oxidative phosphorylation inhibitor sodium azide all markedly reduced sulphate incorporation. These results, together with previous measurements of CO2 production rates, suggest that a functionally significant fraction of the disc's energy is supplied by oxidative phosphorylation. However, low levels of PO2, 2,4-dinitrophenol, and sodium azide have been reported to reduce sulphate incorporation in articular cartilage, a tissue that derives its energy almost entirely from glycolysis.

2,4-Dinitrophenol↗

PubMed1984

Induction of cytoplasmically inherited respiration-deficient ('petite') mutants by photodynamic action of acridine compounds.

All acridines used (acriflavine, proflavine, acridine orange and 3-azido-10-methylacridinium chloride) produced killing in yeast cells when activated with visible light. Acriflavine, proflavine and 3-azido-10-methylacridinium chloride, but not acridine orange, produced petite and sectored colonies. Both cell killing and petite induction by light activation of acriflavine resulted apparently from photodynamic action mediated by singlet oxygen (1O2) since the effect were prevented by either sodium azide or anaerobiosis. The biological effects of 3-azido-10-methylacridinium chloride, which was developed as a potential photoaffinity probe for studying the binding and biological effects of acridines, appeared to be due to a photodynamic action analogous to that of acriflavine. Sodium azide or anaerobiosis prevented the light-activated effects of 3-azido-10-methylacridinium chloride despite the fact that the initial chemical breakdown of the azido derivative induced by light was not affected. Cells suspended in D2O demonstrated an enhanced response to 3-azido-10-methylacridinium chloride with irradiation. These results indicate that singlet oxygen mediates the light-activated biological effects of both acriflavine and 3-azido-10-methylacridinium chloride.

Acridines↗

PubMed1990

Two modes of idiotypic stimulation of T lymphocytes from patients with Chagas' disease: correlations with clinical forms of infection.

Patients with chronic Trypanosoma cruzi infections have peripheral auto-anti-idiotype (Id) T cells that proliferate on exposure to immunoaffinity-purified antibodies against T. cruzi epimastigote antigens (EPI). The responses of some patients' (group 1) peripheral blood mononuclear cells (PBMC) to anti-EPI antibodies from sera of patients with the cardiac form of Chagas' disease (Id-C) were inhibited by chloroquine, but responses of other patients' (group 2) PBMC to Id-C were not inhibited. PBMC responses of both group-1 and -2 patients to anti-EPI antibodies from asymptomatic (indeterminate) patients (Id-I) were inhibited by chloroquine, as were their responses to the antigens in EPI. Most patients (69%) in group 1 had indeterminate Chagas' disease, and 100% of the patients in group 2 had severe, cardiac or digestive Chagas' disease. Both the direct (chloroquine-insensitive) and indirect (processed) modes of stimulation by anti-EPI antibodies required adherent cells. In group 2 (direct stimulation), this requirement was met by exogenous IL-1, and neither anti-HLA-DR,DP(DQ) monoclonal antibody (mAb) nor sodium azide inhibited T-cell proliferation. Indirect Id stimulation of group-1 cells by Id-I or Id-C, and group-2 cells by Id-I or EPI, was inhibited by anti-HLA-DR,DP(DQ) mAb or sodium azide, and exogenous IL-1 alone did not support this processed, MHC-mediated T-cell stimulation, but live adherent cells did. The mode of activation of auto-anti-Id T cells from patients with Chagas' disease depends on the clinical form of infection of both the cell donor and the donor of the stimulating anti-EPI antibodies.

Adult↗

PubMed1981

Mechanism of immune interferon production in vitro: interaction between immune interferon-producing cells and antigenic cells.

Various processes of in vitro immune interferon production by sensitized spleen cells stimulated with allogeneic cells were investigated. When L cells, an interferon-inducing antigen, were fixed with methyl alcohol or paraformaldehyde, the ability to induce immune interferon disappeared. In this immune interferon production system, the majority of sensitized spleen cells adhered to target cells within 1 h of cocultivation. Adherence of immune interferon-producing cells to target cells was observed only when L cell-sensitized spleen cells were cocultured with L cells or with mouse embryo cells derived from C3H mice. Fixation of antigenic cells with methyl alcohol or paraformaldehyde significantly reduced cell adherence. When L cells alone or sensitized spleen cells alone were pretreated separately with cytochalasin D, neither cell type could bind to partner cells. Specific adherence did not take place at 4 degrees C, nor in the presence of dinitrophenol or sodium azide. Continuous protein synthesis in both cells was not required for immune cell adherence. Divalent cations, Ca2+ or Mg2+, were required for this immune specific adherence to take place. However, once stable adherence was established, treatment with cytochalasin D, ethylenediaminetetraacetic acid, or sodium azide, or simple reduction of temperature, did not disrupt the binding. Interaction between immune interferon-producing cells and antigenic cells can be subdivided into two phases according to the requirement for divalent cations: (i) lymphocytes and antigenic cells interact transiently, and divalent cations are required to maintain the binding; (ii) lymphocytes and antigenic cells form a stable interaction, and deprivation of divalent cations does not disrupt the binding. Colchicine showed an inhibitory effect in the period after cell-to-cell adherence. Colchicine did not inhibit the release of interferon. On the other hand, vinblastine, another antimicrotubule agent inhibited the secretion of immune interferon. Since interferon synthesis was not stopped immediately after addition of cycloheximide, continued protein synthesis of sensitized spleen cells was not required for interferon secretion. The present study showed that adherence of immune interferon-producing cells to antigenic cells was a complex phenomenon involving a series of successive events.

Animals↗

PubMed2006

Polycyclic aromatic hydrocarbon (PAH) o-quinones produced by the aldo-keto-reductases (AKRs) generate abasic sites, oxidized pyrimidines, and 8-oxo-dGuo via reactive oxygen species.

Reactive and redox-active polycyclic aromatic hydrocarbon (PAH) o-quinones produced by Aldo-Keto Reductases (AKRs) have the potential to cause depurinating adducts leading to the formation of abasic sites and oxidative base lesions. The aldehyde reactive probe (ARP) was used to detect these lesions in calf thymus DNA treated with three PAH o-quinones (BP-7,8-dione, 7,12-DMBA-3,4-dione, and BA-3,4-dione) in the absence and presence of redox-cycling conditions. In the absence of redox-cycling, a modest amount of abasic sites were detected indicating the formation of a low level of covalent o-quinone depurinating adducts (>3.2 x 10(6) dNs). In the presence of NADPH and CuCl2, the three PAH o-quinones increased the formation of abasic sites due to ROS-derived lesions destabilizing the N-glycosidic bond. The predominant source of AP sites, however, was revealed by coupling the assay with human 8-oxoguanine glycosylase (hOGG1) treatment, showing that 8-oxo-dGuo was the major lesion caused by PAH o-quinones. The levels of 8-oxo-dGuo formation were independently validated by HPLC-ECD analysis. Apyrimidinic sites were also revealed by coupling the assay with Escherichia coli (Endo III) treatment showing that oxidized pyrimidines were formed, but to a lesser extent. Different mechanisms were responsible for the formation of the oxidative lesions depending on whether Cu(II) or Fe(III) was used in the redox-cycling conditions. In the presence of Cu(II)-mediated PAH o-quinone redox-cycling, catalase completely suppressed the formation of the lesions, but mannitol and sodium benzoate were without effect. By contrast, sodium azide, which acts as a *OH and 1O2 scavenger, inhibited the formation of all oxidative lesions, suggesting that the ROS responsible was 1O2. However, in the presence of Fe(III)-mediated PAH o-quinone redox-cycling, the *OH radical scavengers and sodium azide consistently attenuated their formation, indicating that the ROS responsible was *OH.

8-Hydroxy-2'-Deoxyguanosine↗

PubMedDate not supplied

Effect of venom from Cerastes cerastes (Egyptian sand viper) on luminol-dependent chemiluminescence of human blood phagocyte cells.

Cerastes cerastes venom added in vitro to human whole blood caused a marked inhibitory effect on the luminol-dependent chemiluminescence induced by phorbol myristate acetate (PMA) or opsonized zymosan on phagocyte cells. The inhibitory effect produced by the venom was both dose- and time-dependent when PMA was used as the stimulant of the oxidative burst. Similarly, the venom produced a significant inhibitory effect when added to isolated polymorphonuclear leukocytes (PMNs). Incubation of the isolated PMNs with the highest concentration of the venom used (1000 micrograms/ml), however, did not result in any significant disruption of the membranes of these cells. The effect of the venom on the isolated PMNs was also reversible following washing of the venom-treated cells with phosphate-buffered saline. The scavenger of reactive oxygen species such as superoxide dismutase, catalase and dimethyl sulphoxide potentiated the effect of the venom on the luminol-dependent chemiluminescence of human phagocyte cells. Sodium azide, the myeloperoxidase inhibitor, and sodium benzoate produced a similar potentiating effect. The results suggest that some of the toxicity of the venom may be mediated by an action on the phagocytic immune system.

Animals↗

PubMed1987

Differential endocytosis of fluorescein iso-thiocyanate-concanavalin A by normal and chronic myeloid leukemic granulocytes.

Isolated granulocytes from normal individuals and patients suffering from chronic myeloid leukemia (CML) displayed different fluorescent patterns on treatment with fluorescein isothiocyanate concanavalin A (Fl-Con A). The ligand was internalized by 86% of the normal granulocytes, while 80% of the leukemic granulocytes exhibited Fl-Con A localized on the cell periphery. In further experiments, pretreatment of the normal granulocytes with cytochalasin B, iodoacetamide, 2-deoxyglucose and sodium fluoride (but not with sodium azide or dinitrophenol) was found to drastically inhibit internalization of the ligand. However, pretreatment of granulocytes from CML patients with cytochalasin B and 2-deoxyglucose, caused only a little alteration in the pattern of Fl-Con A labelling relative to untreated cells. These results indicate that CML granulocytes are defective in their ability to endocytose Fl-Con A. We suggest that this differential interaction between Fl-Con A and normal and leukemic granulocytes is a convenient system to study the initial steps in receptor mediated endocytosis of Concanavalin A.

Azides↗

PubMed1992

Taurine transport in skate hepatocytes. II. Volume activation, energy, and sulfhydryl dependence.

Isolated skate (Raja erinacea) hepatocytes swollen in hypotonic media exhibited a regulatory volume decrease (RVD) that was associated with only a small increase in K+ or 86Rb+ efflux but a substantial increase in the release of taurine, an amino acid found in high concentrations in skate hepatocytes. Taurine efflux was stimulated in media made hypotonic by addition of H2O or removal of NaCl, as well as in cells swollen in isotonic media containing rapidly penetrating solutes (202 mM ethylene glycol or 202 mM additional urea substituted for 101 mM NaCl), suggesting that cell swelling rather than hyposmolarity is the stimulus for the activation of taurine release. In contrast, release of glutathione, L-[14C]alanine and other alpha-amino acids (e.g., threonine, serine, glutamate, glutamine, glycine, or valine) was unaffected by dilution with 40% H2O. Taurine efflux was not altered by replacement of extracellular Na+ with choline+ or K+ and was only slightly diminished by replacing Cl- with NO3-. Addition of 50 mM taurine or hypotaurine to the incubation media also had no effect on volume-stimulated [14C]taurine efflux, suggesting that the taurine concentration gradient across the plasma membrane is not the driving force. Volume-stimulated taurine transport was temperature sensitive, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibitable (0.5 mM), and nearly completely blocked by metabolic inhibitors (2,4-dinitrophenol, KCN, sodium azide, oligomycin, carbonyl cyanide m-chlorophenylhydrazone, and antimycin A), suggesting an active energy-dependent process. Sulfhydryl-reactive reagents (N-ethylmaleimide, diamide, iodoacetate, tert-butyl hydroperoxide, and mercury) also blocked volume-stimulated taurine efflux, whereas efflux was unaffected by Ca2+ ionophore, phorbol ester, dibutyryl-adenosine 3',5'-cyclic monophosphate, vasopressin, or pretreatment with ouabain or furosemide. N-ethylmaleimide, diamide, 2,4-dinitrophenol, and iodoacetate plus KCN also inhibited the RVD. These findings suggest that, in contrast to hepatocytes from most vertebrate species, RVD in skate hepatocytes is associated with the release of only a small fraction of intracellular K+ but a substantial fraction of intracellular taurine and perhaps other organic osmolytes. This volume-activated taurine transport mechanism is energy and sulfhydryl group dependent and is not related to the taurine concentration gradient across the skate hepatocyte plasma membrane.

Amino Acids↗

PubMed2004

Fluorescence resonance energy transfer from tryptophan to a chromium(III) complex accompanied by non-specific cleavage of albumin: a step forward towards the development of a novel photoprotease.

A chromium(III) complex, transdiaqua [N, N'-propylenebis(salicylideneimino)chromium(III)]perchlorate ([Cr(salprn)(H2O)(2)]ClO(4)) in the presence of sodium azide and upon photoexcitation was found to bring about non-selective cleavage of bovine serum albumin (BSA). Electron paramagnetic resonance (EPR) evidence has been obtained for the formation of a Cr(V) species upon photolysis of a solution containing the chromium(III) complex and sodium azide. This Cr(V) species non-selectively cleaves BSA. The fluorescence excitation spectrum of BSA-[Cr(salprn)(H2O)(2)]+ adduct showed a band at lambda(max)(ex) nm due to charge transfer transition of the chromium(III) complex as well as a prominent band at 290 nm attributable to tryptophan absorption. This indicated an efficient Forster type fluorescence energy transfer (FRET) from the tryptophan residues to the chromium(III) complex indicating that the Cr(III) complex binds in the vicinity of the tryptophan residue.

Animals↗

PubMed1992

Zinc transport into endothelial cells is a facilitated process.

The kinetics of zinc transport were examined by measuring the uptake of 65Zn into cultured endothelial cells. This served as a suitable model for characterizing the transport of zinc across a biological membrane (i.e., the plasma membrane). The transport process was saturable under physiological conditions, which indicates a facilitating transport mechanism. Within the physiological range of zinc concentrations, the maximum zinc transport rate was 27 pmoles zinc/(min x mg protein) and it was half maximal at 4.1 microM zinc. Cadmium competitively inhibited zinc transport (Ki = 6.5 microM), while equimolar concentrations of copper and manganese were ineffectual. The rate of zinc transport was substantially reduced at lower temperatures and in the presence of sulfhydryl blockers (sodium iodoacetate and N-ethylmaleimide). Inhibitors of energy metabolism (2,4-dinitrophenol and sodium azide) failed to disrupt zinc transport. These results demonstrate that zinc transport into endothelial cells is a facilitated process (i.e., it is carrier mediated and energy-independent).

2,4-Dinitrophenol↗

PubMed1991

Isolation of 34 kDa protein from spinach chloroplasts having ferrooxidase and H2O2-dependent dark O2 evolution activities.

A latent form of 'Ferrooxidase' exhibiting ferrocyanide-dependent O2 uptake was detected in the isolated spinach chloroplasts. Presence of a cationic detergent hexadecyl trimethyl ammonium bromide (CTAB) in the medium was essential to induce this activity. The association of this enzyme activity with photosystem II (PSII) particles as well as the ability of PSII particles to show oxidation of H2O2 (catalase like activity) indicated its possible relationship with water oxidation system. The protein catalysing this activity was purified to homogeneity and its molecular mass was found to be 34 kDa. The purified protein showed a complete dependence on an electron acceptor, namely ferricyanide, for the oxidation of H2O2. While with ferrocyanide in the presence of CTAB, the protein exhibits the ferrooxidase activity. For both activities, a sharp pH optima at 6.1 was observed. The km for H2O2 was 12.2 mM. The purified enzyme protein contained 4 atoms of calcium and 2 atoms of iron per mole of the enzyme. Unlike catalase, the enzyme reaction was insensitive to sodium azide even at 500 microM concentration. The enzyme was found to be sensitive to metal chelators like ethylene-glycol-bis-(beta-aminoethylether) N, N+ tetra acetic acid (EGTA) (2mM), alpha,alpha-dipyridyl (500 microM) and 1,10-orthophenanthroline (200 microM). The sensitivity of the reaction to alpha,alpha-dipyridyl and 1,10-orthophenanthroline suggested the involvement of Fe2+ in the reaction. Inhibition of enzyme activity by EGTA and restoration of activity by supplementation of CaCl2 to the EGTA-dialysed sample confirmed the absolute requirement for calcium for this activity. Calcium was absent in the EGTA-dialysed enzyme. Apart from these inhibitors, NaF and NH2OH were potent inhibitors of the enzyme reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Azides↗

PubMed2006

The effects of biocides (antiseptics and disinfectants) on the endospores of Rhinosporidium seeberi.

No data exists on the activity of biocides (antiseptics and disinfectants) on Rhinosporidium seeberi that causes rhinosporidiosis in humans and animals. On account of the inability to culture R. seeberi, in vitro, dyes were used to assess the morphological integrity and viability of biocide-treated endospores that are considered to be the infective stage of this pathogen. Evan's Blue (EvB) identifies the morphological integrity of the endospores while MTT (3-[4, 5-dimethylthiazol-2yl]-2, 5-diphenyl tetrazolium bromide) identifies metabolic activity through its reduction by cellular dehydrogenases to microscopically visible deposits of insoluble formazan. MTT-negativity has earlier been shown to correlate with absence of growth of yeast and mycelial fungi in culture and could thus indicate the loss of viability of MTT-negative rhinosporidial endospores. Hydrogen peroxide, glutaraldehyde, chloroxylenol, chlorhexidine, cetrimide, thimerosal, 70% ethanol, iodine in 70% ethanol, 10% formalin, povidone-iodine, sodium azide and silver nitrate were tested on freshly-harvested endospores and all biocides caused metabolic inactivation with or without altered structural integrity as shown by absence of MTT-staining after 3, 24 or 36 hour after exposure, while EvB stained only the endospores treated with sodium azide, ethanol, thimerosal, chloroxylenol, glutaraldehyde and hydrogen peroxide. With clinically useful biocides - chlorhexidine, cetrimide-chlorhexidine, 70% ethanol, povidone-iodine and silver nitrate, a total period of exposure of endospores to the biocide, for seven minutes, produced metabolic inactivation of the endospores. Anti-rhinosporidial antiseptics that could be used in surgery on rhinosporidial patients include povidone-iodine in nasal packs for nasal and naso-pharyngeal surgery, chlorhexidine and cetrimide-chlorhexidine on the skin, while povidone-iodine and silver nitrate could have application in ocular rhinosporidiosis.

Animals↗

PubMedDate not supplied

Two- and three-dimensional distributions of opioid receptors on NG108-15 cells visualized with the aid of fluorescence confocal microscopy and anti-idiotypic antibodies.

Polyclonal anti-idiotypic antibodies previously shown to be specific for mu- and delta-opioid receptor subclasses on rat brain membrane preparations using radioreceptor assays have been labeled with fluorescent conjugates of avidin. The resulting complexes have been used to study the distribution and properties of opioid receptors on living and fixed NG108-15 cells using normal fluorescence, and confocal fluorescence, microscopy. From the confocal data three-dimensional views of immunofluorescent distributions have been obtained using a computer transformation of the data. From experiments involving co-incubation with media containing naloxone, selective opioid peptides and stereoisomeric synthetic opioid analogs as blocking agents, the specificity and stereospecificity of immunofluorescent binding is reported. The effects on immunofluorescent binding by co-incubation with media containing a sulfhydryl reagent, 100 mM-NaCl and sodium azide are also reported. The results have implications concerning the use of immunoprecipitation in the biochemical isolation of the receptors. Finally, the confocal two-dimensional sections and two-dimensional reconstructions obtained from fixed cells show internal immunofluorescence in the form of cytoplasmic clusters. The reagents and methods should be applicable to visualizing native opioid receptor distributions on neurons.

Animals↗

PubMed2003

DNA is a target of the photodynamic effects elicited in A2E-laden RPE by blue-light illumination.

PURPOSE: When the pyridinium bisretinoid A2E, an age-related fluorophore in the retinal pigment epithelium (RPE), is irradiated with blue light, photochemical events are initiated that can ultimately provoke cell death. This study was designed to determine whether DNA is a target of the cellular damage. METHODS: ARPE-19 cells accumulated A2E before exposure to blue light. DNA damage was assayed in individual cells by alkaline gel electrophoresis (comet assay), with and without the addition of the repair enzymes formamidopyrimidine N-glycosylase (Fpg), endonuclease III (endo III) and T4-endonuclease V (T4-endo V) to characterize DNA lesions. Damage was quantified as comet tail moment. The base lesion 8-oxo-deoxyguanosine (8-oxo-dG) was detected by immunoperoxidase and histochemical methods. The singlet oxygen quencher, sodium azide, was tested for its ability to reduce DNA damage, and cell viability was quantified. RESULTS: DNA damage was induced in A2E-containing RPE exposed to 430-nm illumination. The extent of damage, measured as tail moment, was proportional to exposure duration and was reduced by preincubation with sodium azide. The detection of FPG- and endo III-sensitive DNA lesions revealed the presence of oxidized purine and pyrimidine bases, whereas labeling with specific antibody and binding of fluorescein-labeled avidin indicated that guanine bases were oxidatively modified to 8-oxo-dG. The ability of the cells to repair the DNA damage declined as the severity was increased, and kinetic studies disclosed rapid and slow stages of repair. CONCLUSIONS: DNA is one of the cellular constitutents that can be damaged by the interaction of A2E and blue light. At least some of the DNA lesions are oxidative base modifications.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1975

Cytochemical localization of peroxidase activity in the miracidium of Schistosoma mansoni.

Distribution of peroxidase activity in the mitochondria of the miracidium of the blood fluke, Schistosoma mansoni, was investigated cytochemically using the diaminobenzidine (DAB) technique. Re-action product was localized in the mitochondria of this larvae stage at pH 7.4 and 9.7. The reaction was peroxide-dependent and insensitive to either potassium cyanide, sodium azide, or 3-amino-1,2,4-triazole at the concentrations used. The reaction was inactivated by heat and by pretreatment with methanol-nitro-ferricyanide, and inhibitor of peroxidase. A perioxide-independent reaction was also observed in the mitochondria. This latter reaction was sensitive to potassium cyanide and sodium azide. It is hypothesized that the peroxidase either may act where peroxide is an electron acceptor in a flavoprotein-linked system or may be a vestige of a more primitive pathway. No peroxidase activity was observed in the mitochondria of other stages of the life cycle of the worm.

Animals↗

PubMed1979

Sugar transport in Coprinus cinereus.

Two transport systems for glucose were detected: a high affinity system with a Km of 27 muM, and a low affinity system with a Km of 3.3 mM. The high affinity system transported glucose, 2-deoxy-D-glucose (Km = 26 muM), 3-O-methylglucose (Km = 19 muM), D-glucosamine (Km = 652 muM), D-fructose (Km = 2.3 mM) and L-sorbose (Km = 2.2 mM). All sugars were accumulated against concentration gradients. The high affinity system was strongly or completely inhibited by N-ethylmaleimide, quercetin, 2,4-dinitrophenol and sodium azide. The system had a distinct pH optimum (7.4) and optimum temperature (45 degrees C). The low affinity system transported glucose, 2-deoxy-D-glucose (Km = 7.5 mM), and 3-O-methylglucose (Km = 1.5 mM). Accumulation again occurred against a concentration gradient. The low affinity system was inhibited by N-ethylmaleimide, quercetin and 2,4-dinitrophenol, but not by sodium azide. The rate of uptake by the low affinity system was constant over a wide temperature range (30--50 degrees C) and was not much affected by pH; but as the pH of the medium was altered from 4.5 to 8.9 a co-ordinated increase in affinity for 2-deoxy-D-glucose (from 52.1 mM to 0.3 mM) and decrease in maximum velocity (by a factor of five) occurred. Both uptake systems were present insporelings germinated in media containing sodium acetate as sole carbon source. Only the low affinity system could initially be demonstrated in glucose-grown tissue, although the high affinity system was restored by starvation inglucose-free medium. The half-ti me for restoration of high affinity activity was 3.5 min and the process was unaffected by cycloheximide. Addition of glucose to an acetate-grown culture inactivated the high affinity system with a half-life of 5--7.5 s. Addition of cycloheximide to an acetate-grown culture caused decay of the high affinity system with a half-life of 80 min. Regulation is thus thought to depend on modulation of protein activity rather than synthesis, and the kinetics of glucose, 2-deoxy-D-glucose and 3-O-methylglucose uptake would be consistent with there being a single carrier showing negative co-operativity. Analysis of transport defective mutants revealed defects in both transport systems although the mutants used were alleles of a single gene. It is concluded that this gene (the ftr cistron) is the structural gene for an allosteric molecule which serves both transport systems.

Agaricales↗