A superoxide dismutase purified from the roots from Stemona tuberosa.

Proteins from the fresh roots of Stemona tuberosa (Stemonaceae) were extracted into 20 mM phosphate buffer, pH 7.2/0.1 M NaCl, precipitated with 90% saturation ammonium sulfate, and enriched by diethylaminoethanol (DEAE) cellulose. The protein eluted as a single main peak from the unbound fractions (ST-1), and appeared as a single band with superoxide dismutase (SOD) activity after native polyacrylamide gel electrophoresis (PAGE) resolution and zymogram development. ST-1 was classified as SOD due to its strong inhibition by HCN and H2O2. The amino acid sequence of three tryptic peptides of ST-1 matched with the SOD isozymes from Ananas comosus and Solanum lycopersicum. The SOD consisted of at least two heterologous protein subunits with molecular mass of 17.6 and 31.5 kD, respectively, and had an optimal SOD activity at pH 5 and over a temperature range of 0-50°C. MgCl2, MnCl2, and HgCl2 were strongly inhibitory at all concentrations tested. The SOD activity was completely negated in the presence of 0.5 mM SDS or 5 mM HgCl2. The relationship between riboflavin and nitroblue tetrazolium (NBT) on SOD activity was linear, giving K m and V max values of the purified SOD of 62.414 ± 0.015 M and 101.010 ± 0.022 µmol/min/mg protein for NBT and 27.389 ± 0.032 M and 38.167 ± 0.021 µmol/min/mg protein for riboflavin, respectively.

Effect of storage on the nitro blue tetrazolium reduction test in dog blood samples.

INTRODUCTION: The nitro blue tetrazolium (NBT) reduction test (NBTT) has been used for measuring the metabolic activity of phagocytes of mammals. Activated neutrophils transform NBT into formazan in the cytoplasm. The NBTT can detect the activation of neutrophils in peripheral blood and is used to assess neutrophil function in dogs. However, the NBTT is not used frequently in the clinical setting, as samples should be processed after blood collection. OBJECTIVE: The aim of this study was to evaluate the effect of storage on NBTT in dog blood samples. MATERIALS AND METHODS: Residual EDTA blood samples from 22 dogs were included of different ages, breeds, and sex. The buffy coat layer was separated from the blood and incubated with 0.1% NBT. The NBTT was performed at 0, 24, 48, and 72 h after the collection of blood. Blood samples were stored at 4°C until the tests were performed. Blood smears were evaluated by light microscopy, and the NBT reduction rate was reported, which represents the percentage of activated neutrophils. The NBT reduction rate was calculated after counting 300 neutrophils in each slide. RESULTS: The means of NBTT in dog blood samples at 0, 24, 48, and 72 h were 8.3%, 8.5%, 8.7%, and 7.8%, respectively. No significant differences were observed between time points. CONCLUSIONS: This study showed that the NBTT can be performed up to 72 h after the collection of canine blood if correctly refrigerated at 4°C. This finding supports the performance of the NBTT in the clinical setting.

NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation.

NOX4 is an enigmatic member of the NOX (NADPH oxidase) family of ROS (reactive oxygen species)-generating NADPH oxidases. NOX4 has a wide tissue distribution, but the physiological function and activation mechanisms are largely unknown, and its pharmacology is poorly understood. We have generated cell lines expressing NOX4 upon tetracycline induction. Tetracycline induced a rapid increase in NOX4 mRNA (1 h) followed closely (2 h) by a release of ROS. Upon tetracycline withdrawal, NOX4 mRNA levels and ROS release decreased rapidly (<24 h). In membrane preparations, NOX4 activity was selective for NADPH over NADH and did not require the addition of cytosol. The pharmacological profile of NOX4 was distinct from other NOX isoforms: DPI (diphenyleneiodonium chloride) and thioridazine inhibited the enzyme efficiently, whereas apocynin and gliotoxin did not (IC(50)>100 muM). The pattern of NOX4-dependent ROS generation was unique: (i) ROS release upon NOX4 induction was spontaneous without need for a stimulus, and (ii) the type of ROS released from NOX4-expressing cells was H(2)O(2), whereas superoxide (O(2)(-)) was almost undetectable. Probes that allow detection of intracellular O(2)(-) generation yielded differential results: DHE (dihydroethidium) fluorescence and ACP (1-acetoxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine) ESR measurements did not detect any NOX4 signal, whereas a robust signal was observed with NBT. Thus NOX4 probably generates O(2)(-) within an intracellular compartment that is accessible to NBT (Nitro Blue Tetrazolium), but not to DHE or ACP. In conclusion, NOX4 has a distinct pharmacology and pattern of ROS generation. The close correlation between NOX4 mRNA and ROS generation might hint towards a function as an inducible NOX isoform.

H2O2 release from human granulocytes during phagocytosis. Relationship to superoxide anion formation and cellular catabolism of H2O2: studies with normal and cytochalasin B-treated cells.

Normal and cytochalasin B-treated human granulocytes have been studied to determine some of the interrelationships between phagocytosis-induced respiration and superoxide and hydrogen peroxide formation and release into the extracellular medium by intact cells. By using the scopoletin fluorescent assay to continuously monitor extracellular hydrogen peroxide concentrations during contact of cells with opsonized staphylococci, it was demonstrated that the superoxide scavengers ferricytochrome c and nitroblue tetrazolium significantly reduced the amount of H(2)O(2) released with time from normal cells but did not abolish it. This inhibitory effect was reversed by the simultaneous addition of superoxide dismutase (SOD), whereas the addition of SOD alone increased the amount of detectable H(2)O(2) in the medium. The addition of sodium azide markedly inhibited myeloperoxidase-H(2)O(2)-dependent protein iodination and more than doubled H(2)O(2) release, including the residual amount remaining after exposure of the cells to ferricytochrome c, suggesting its origin from an intracellular pool shared by several pathways for H(2)O(2) catabolism. When cells were pretreated with cytochalasin B and opsonized bacteria added, reduced oxygen consumption was observed, but this was in parallel to a reduction in specific binding of organisms to the cells when compared to normal. Under the influence of inhibited phagosome formation by cytochalasin B, the cells released an increased amount of superoxide and peroxide into the extracellular medium relative to oxygen consumption, and all detectable peroxide release could be inhibited by the addition of ferricytochrome c. Decreased H(2)O(2) production in the presence of this compound could not be ascribed to diminished bacterial binding, decreased oxidase activity, or increased H(2)O(2) catabolism and was reversed by the simultaneous addition of SOD. Furthermore, SOD and ferricytochrome c had similar effects on another H(2)O(2)-dependent reaction, protein iodination, in both normal and cytochalasin B cells. When oxygen consumption, O(2.) (-), and H(2)O(2) release were compared in the presence of azide under identical incubation conditions, the molar relationships for normal cells were 1.00:0.34:0.51 and for cytochalasin B-treated cells 1.00:0.99:0.40, respectively. Nonopsonized, or opsonized but disrupted, bacteria did not stimulate any of these metabolic functions. The results indicate that with normal cells approximately 50% of H(2)O(2) released during phagocytosis is derived directly from O(2.) (-) by dismutation, the remainder appearing from an (intra)cellular source shared by azide-inhibitable heme enzymes. With cytochalasin B treatment the evidence is consistent with the derivation of all H(2)O(2) from an O(2.) (-) precursor which is released from the cell surface. Furthermore, when activated by phagocytic particle binding, the neutrophil O(2.) (-) generating system appears to make more of this compound than can be accounted for by dismutation to H(2)O(2). This establishes conditions for the direct participation of both compounds in the microbicidal and cytocidal activity of these cells.

Differential effects of peroxynitrite on the function of arginine vasopressin V(1a) receptors and alpha(1)-adrenoceptors in vivo.

OBJECTIVE: The aim of this study was to provide evidence that peroxynitrite may differentially affect the function of arginine vasopressin (AVP) V(1a) receptors and alpha(1)-adrenoceptors in vascular smooth muscle of the rat METHODS: The vasoconstrictor responses elicited by AVP, or the alpha(1)-adrenoceptor agonist, phenylephrine, were determined in anesthetized rats before and after injections of (i) peroxynitrite, the thiol chelator, para-hydroxymercurobenzoic acid (PHMBA), or the electron acceptor, nitroblue tetrazolium (NBT). The ability of the reducing agent, glutathione, to reverse the loss of response to phenylephrine and AVP in peroxynitrite-treated rats was also examined. RESULTS: The AVP-induced responses were suppressed 10-20 min but not 60-70 min after the administration of peroxynitrite. Glutathione reversed the above loss of response to AVP at 10-20 min. The responses elicited by phenylephrine were suppressed 10-20 min and 60-70 min after administration of peroxynitrite. Glutathione did not reverse the above losses of response to phenylephrine. In addition, the vasoconstrictor actions of AVP and phenylephrine were markedly suppressed after administration of PHMBA or nitroblue tetrazolium. CONCLUSIONS: The above findings provide evidence that exogenously administered peroxynitrite may differentially affect the function of AVP V(1a) receptors and alpha(1)-adrenoceptors in vascular smooth muscle of the rat. The possibility that peroxynitrite impairs AVP V(1a) receptor function by transient oxidation events whereas peroxynitrite impairs alpha(1)-adrenoceptor function by transient oxidation and permanent nitration events will be discussed.

Hypoxic vasoconstriction of partial muscular intra-acinar pulmonary arteries in murine precision cut lung slices.

BACKGROUND: Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) which serves to match lung perfusion to ventilation. The underlying mechanisms are not fully resolved yet. The major vascular segment contributing to HPV, the intra-acinar artery, is mostly located in that part of the lung that cannot be selectively reached by the presently available techniques, e.g. hemodynamic studies of isolated perfused lungs, recordings from dissected proximal arterial segments or analysis of subpleural vessels. The aim of the present study was to establish a model which allows the investigation of HPV and its underlying mechanisms in small intra-acinar arteries. METHODS: Intra-acinar arteries of the mouse lung were studied in 200 mum thick precision-cut lung slices (PCLS). The organisation of the muscle coat of these vessels was characterized by alpha-smooth muscle actin immunohistochemistry. Basic features of intra-acinar HPV were characterized, and then the impact of reactive oxygen species (ROS) scavengers, inhibitors of the respiratory chain and Krebs cycle metabolites was analysed. RESULTS: Intra-acinar arteries are equipped with a discontinuous spiral of alpha-smooth muscle actin-immunoreactive cells. They exhibit a monophasic HPV (medium gassed with 1% O2) that started to fade after 40 min and was lost after 80 min. This HPV, but not vasoconstriction induced by the thromboxane analogue U46619, was effectively blocked by nitro blue tetrazolium and diphenyleniodonium, indicating the involvement of ROS and flavoproteins. Inhibition of mitochondrial complexes II (3-nitropropionic acid, thenoyltrifluoroacetone) and III (antimycin A) specifically interfered with HPV, whereas blockade of complex IV (sodium azide) unspecifically inhibited both HPV and U46619-induced constriction. Succinate blocked HPV whereas fumarate had minor effects on vasoconstriction. CONCLUSION: This study establishes the first model for investigation of basic characteristics of HPV directly in intra-acinar murine pulmonary vessels. The data are consistent with a critical involvement of ROS, flavoproteins, and of mitochondrial complexes II and III in intra-acinar HPV. In view of the lack of specificity of any of the classical inhibitors used in such types of experiments, validation awaits the use of appropriate knockout strains and siRNA interference, for which the present model represents a well-suited approach.

Differential effects of nitroblue tetrazolium on the hemodynamic responses elicited by activation of alpha1-adrenoceptors and 5-HT2 receptors in conscious rats.

This study determined the effects of the lipophobic electron acceptor, nitroblue tetrazolium (5 micromol/kg, i.v.) on the vasoconstrictor responses elicited by the 5-hydroxytryptamine2 (5-HT2) receptor agonist, alpha-methyl-5-HT (5-50 microg/kg, i.v.) and the alpha1-adrenoceptor agonist, phenylephrine (2.5-20 microg/kg, i.v.) in conscious Sprague-Dawley rats. The systemic injection of nitroblue tetrazolium elicited pronounced hemodynamic responses that subsided by 10-15 min. Prior to the administration of nitroblue tetrazolium, the injections of alpha-methyl-5-HT and phenylephrine elicited dose-dependent increases in mean arterial blood pressure and mesenteric, renal and hindquarter vascular resistances. After administration of nitroblue tetrazolium, the vasoconstrictor responses elicited by alpha-methyl-5-HT were augmented whereas those elicited by phenylephrine were diminished. These results are consistent with the possibility that nitroblue tetrazolium interacts with the extracellular ligand-binding domains of 5-HT2 receptors and alpha1-adrenoceptor and that this interaction has opposite effects on activities of these G protein coupled receptors.

A fast and easy nitroblue tetrazolium method for carrier screening and prenatal detection of chronic granulomatous disease.

BACKGROUND: Analysis of the functional activity of neutrophils is of great importance in the differential diagnosis of patients with recurrent bacterial infections. It has long been established that stimulated polymorphonuclear leukocytes reduce nitroblue tetrazolium. Application of a simple and reliable nitroblue tetrazolium method that clearly differentiates the chronic granulomatous disease patients and heterozygote carriers in some groups suspected to have chronic granulomatous disease was investigated. METHODS: This study consisted of 197 samples taken from 100 children (2 - 24-month-old) and 81 neonates (aged < 2 months) referred to our center either due to a suspected bacterial infection or suspected immunodeficiency. The sample also included 16 cord blood samples. Fifty healthy adult individuals were enrolled in this study and were diagnosed as normal control. Neutrophil reduction of nitroblue tetrazolium can be stimulated in vitro by protein kinase agonists such as phorbol myristate acetate, resulting in release of superoxide anion. RESULTS: Phorbol myristate acetate is an exceptionally powerful stimulant and when used in conjunction with glass adherence, caused nearly all normal neutrophils to become transformed and reduced nitroblue tetrazolium to formazan deposits. Of 197 blood samples, 9 were diagnosed as having unrelated chronic granulomatous disease and 7 were carriers of X-linked or autosomal recessive chronic granulomatous disease. The carriers had a range of 15 - 75% stimulated neutrophils. CONCLUSION: We have established a phorbol myristate acetate-stimulated nitroblue tetrazolium test for detection of chronic granulomatous disease patients, which clearly differentiates the chronic granulomatous disease patients from heterozygote carriers. The results in cord fetal blood indicate that this test may be used for antenatal diagnosis of affected boys, carrier females, and autosomal recessive variants of chronic granulomatous disease. The technique is simple, fast, inexpensive, and requires only a few microliters of blood.

Studies on the lysyl hydroxylase reaction. II. Inhibition kinetics and the reaction mechanism.

Product inhibition of lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) was studied with succinate, CO2, dehydroascorbate and hydroxylysine-rich polypeptide chains. The product inhibition patterns and addition data are consistent with a reaction mechanism involving an ordered binding of Fe2+, alpha-ketoglutarate, O2 and the peptide substrate to the enzyme in this order, and an ordered release of the hydroxylated peptide, CO2, succinate and Fe2+, in which Fe2+ need not leave the enzyme during each catalytic cycle and in which the order of release of the hydroxylated peptide and CO2 is uncertain. Ascorbate probably reacts by a substitution mechanism, either after the release of the hydroxylated peptide, CO2 and succinate or after the release of all products, including Fe2+, and dehydroascorbate is released before the binding of Fe2+. It is suggested that the ascorbate reaction is required to reduce either the enzyme-iron complex or the free enzyme, which may be oxidized by a side-reaction during some catalytic cycles, but not the majority. The mechanisms of the prolyl 4-hydroxylase and lysyl hydroxylase reactions are suggested to be identical. Zn2+, several citric acid cycle intermediates, nitroblue tetrazolium and homogentisic acid inhibited lysyl hydroxylase competitively with regard to Fe2+, alpha-ketoglutarate, O2 and ascorbate respectively, and epinephrine non-competitively with regard to all cosubstrates. Apparent Ki values are given for the product and other inhibitors.

Redox activation of intracellular calcium release channels (ryanodine receptors) in the sustained phase of hypoxia-induced pulmonary vasoconstriction.

Hypoxia-induced pulmonary vasoconstriction (HPV) is an important adaptive process that remains incompletely understood. In preconstricted rat pulmonary arteries (inner diameter, 250 to 400 microm), hypoxia (pO2 approximately 10 mm Hg) induces an initial transient phase and a more slowly developing sustained phase of vasoconstriction. Since the release of calcium ions (Ca2+) from intracellular stores by redox-sensitive intracellular Ca2+ release channels known as ryanodine receptors (RyRs) in pulmonary arterial smooth-muscle cells (PASMCs) may play a role in HPV, and considerable evidence now supports that levels of reactive oxygen species (ROS) are paradoxically increased in PASMC under hypoxia, we investigated whether redox activation of RyRs by ROS may transduce HPV. By reverse transcriptase-polymerase chain reaction, we found that all three RyR isoforms are expressed in rat pulmonary arteries and in PASMCs. The sustained phase, but not the transient phase, of HPV can be prevented by pretreating pulmonary arteries with RyR inhibitors ryanodine (200 micromol/L) or dantrolene (50 micromol/L). The addition of dantrolene, ryanodine or the thiol-reducing agent dithiothreitol (1 mmol/L) during the sustained phase of HPV reversed the hypoxic vasoconstriction. In contrast, the superoxide scavenger nitroblue tetrazolium (500 nmol/L) prevented further hypoxic pulmonary vasoconstriction during the sustained phase of HPV but did not reverse it. Taken together, our data suggest that redox activation of RyRs by ROS has an important role in transducing the sustained contraction of pulmonary arteries under hypoxia.

Potential role of nitration and oxidation reactions in the effects of peroxynitrite on the function of beta-adrenoceptor sub-types in the rat.

This study examined the hemodynamic responses elicited by the beta-adrenoceptor agonist, isoproterenol (1 and 10 microg/kg, i.v.) before and after administration of (i) peroxynitrite (10 x 10 micromol/kg, i.v.), (ii) the thiol chelator, para-hydroxymercurobenzoic acid (pHMBA, 75 micromol/kg, i.v.), and (iii) the electron acceptor, nitroblue tetrazolium (NBT, 10 micromol/kg, i.v.) in pentobarbital-anesthetized rats. The tachycardia elicited by the lower dose of isoproterenol was diminished whereas the tachycardia elicited by the higher dose was not attenuated after administration of peroxynitrite. The falls in hindquarter and renal vascular resistances elicited by both doses of isoproterenol were substantially diminished whereas the isoproterenol-induced falls in mesenteric vascular resistance were not changed after administration of peroxynitrite. All of the isoproterenol-induced responses were markedly attenuated after administration of pHMBA or NBT. These findings suggest that the oxidation and/or nitration of beta-adrenoceptors impair the ability of isoproterenol to bind to and/or activate these G protein-coupled receptors. beta1-, beta2- and beta3-adrenoceptors contain extracellular cysteine residues susceptible to oxidation (i.e., disulfide-bridge formation) whereas only the beta1- and beta2-adrenoceptors contain extracellular tyrosine residues susceptible to nitration. These findings also suggest that sustained impairment of beta1- and beta2-adrenoceptor function by peroxynitrite is due to nitration of extracellular tyrosine residues in these receptors. By analogy, beta3-adrenoceptors may not be permanently affected by peroxynitrite because these receptors are devoid of extracellular tyrosine residues.

Caloric restriction augments ROS defense in S. cerevisiae, by a Sir2p independent mechanism.

Aging is associated with increased production of reactive oxygen species (ROS) and oxidation-induced damage to intracellular structures and membranes. Caloric restriction (CR) has been demonstrated to delay aging in a variety of species. Although the mechanisms of CR remain to be clearly elucidated, reductions in oxidative damage have been shown to increase lifespan in several model systems. Contrary to the general belief that ROS production is reduced in CR, this article provides evidence that not only oxygen consumption but ROS production is enhanced in the calorie restricted condition. To understand the biological mechanism underlying the anti aging action of CR, the role of scavenging enzymes was studied. It was found that super oxide dismutase (SOD1 and SOD2), catalase and glutathione peroxidase (GPx) all are over expressed in CR. We further investigated the role of Sir2, a potential effector of CR response in the activation of scavenging enzymes. No marked difference was found in CR mediated activation of SOD and catalase in the absence of Sir2. Our results suggest that in CR scavenging enzymes are activated by a Sir2 independent manner.

Mechanism, measurement, and prevention of oxidative stress in male reproductive physiology.

Numerous factors influence male fertility. Among these factors is oxidative stress (OS), which has elicited an enormous interest in researchers in recent period. Reactive oxygen species (ROS) are continuously produced by various metabolic and physiologic processes. OS occurs when the delicate balance between the production of ROS and the inherent antioxidant capacity of the organism is distorted. Spermatozoa are particularly sensitive to ROS as their plasma membrane contains polyunsaturated fatty acids (PUFA), which oxidizes easily. They also lack cytoplasm to generate a robust preventive and repair mechanism against ROS. The transition metal ions that are found in the body have a catalytic effect in the generation of ROS. Lifestyle behaviours such as smoking and alcohol use and environmental pollution further enhance the generation of ROS and thus, cause destructive effects on various cellular organelles like mitochondria, sperm DNA etc. This article analyzes the detrimental effects of OS on male fertility, measurement of OS and effective ways to decrease or eliminate them completely. We have also provided information on oxidative stress in other systems of the body, which may be applied to future research in the field of reproductive biology.

Dopa decarboxylase exhibits low pH half-transaminase and high pH oxidative deaminase activities toward serotonin (5-hydroxytryptamine).

Dopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but also side reactions including half-transamination of D-aromatic amino acids and oxidative deamination of aromatic amines. The latter reaction produces, in equivalent amounts, an aromatic aldehyde or ketone (depending on the nature of the substrate), and ammonia, accompanied by O(2) consumption in a 1 : 2 molar ratio with respect to the products. The kinetic mechanism and the pH dependence of the kinetic parameters have been determined in order to obtain information on the chemical mechanism for this reaction toward 5-hydroxytryptamine (5-HT). The initial velocity studies indicate that 5-HT and O(2) bind to the enzyme sequentially, and that D-Dopa is a competitive inhibitor versus 5-HT and a noncompetitive inhibitor versus O(2). The results are consistent with a mechanism in which 5-HT binds to DDC before O(2). The pH dependency of log V for the oxidative deaminase reaction shows that the enzyme possesses a single ionizing group with a pK value of approximately 7.8 that must be unprotonated for catalysis. In addition to an ionizing residue with a pK value of 7.9 similar to that found in the V profile, the (V/K)(5-HT) profile exhibits a pK value of 9.8, identical to that of free substrate. This pK was therefore tentatively assigned to the alpha-amino group of 5-HT. No titratable ionizing residue was detected in the (V/K)(O2) profile, in the pH range examined. Surprisingly, at pH values lower than 7, where oxidative deamination does not occur to a significant extent, a half-transamination of 5-HT takes place. The rate constant of pyridoxamine 5'-phosphate formation increases below a single pK of approximately 6.7. This value mirrors the spectrophotometric pK(spec) of the shift 420-384 nm of the external aldimine between DDC and 5-HT. Nevertheless, the analysis of the reaction of DDC with 5-HT under anaerobic conditions indicates that only half-transamination occurs with a pH-independent rate constant over the pH range 6-8.5. A model accounting for these data is proposed that provides alternative pathways leading to oxidative deamination or half-transamination.

Nod factor induction of reactive oxygen species production is correlated with expression of the early nodulin gene rip1 in Medicago truncatula.

Plant genes that are specifically activated by the rhizobial lipochitooligosaccharide signal molecule (Nod factor) in legume hosts are collectively referred to as nodulins. Although nodulin gene expression is both spatially and temporally correlated with symbiosis, the function of these genes and the molecular events underlying their expression remain unknown. Sequence analysis of rip1, an early nodulin gene encoding a putative peroxidase protein, revealed the existence of sequence motifs with homology to reactive oxygen species (ROS) responsive cis elements. Here we report that recognition of compatible Nod factor rapidly stimulates a spatially localized production of reactive oxygen species in legume roots. Sinorhizobium meliloti mutants that produce an altered Nod factor structure and a nonnodulating plant mutant, dmi1-1, that is implicated in Nod factor signal transduction are equally impaired in the ability to elicit ROS production and rip1 expression. Interestingly, both rip1 transcription and ROS production exhibit the same tissue-specific pattern of localization. Moreover, exogenous hydrogen peroxide is sufficient to activate rip1 transcription. Taken together, these results suggest that ROS production is a consequence of specific Nod factor perception and implicate H2O2 produced during this response as a mediator of Nod factor-induced rip1 expression.

Electron spin resonance detection of oxygen-centred radicals in murine macrophages stimulated with bacterial endotoxin.

The production of oxygen radicals by Bacille-Calmette-Guerin primed mouse macrophages stimulated with bacterial endotoxin has been investigated. Superoxide radicals were spin-trapped in this system with dimethylpyrroline-N-oxide after a lag period of 20-40 minutes. The electron spin resonance signals due to the superoxide radical adduct could be inhibited by superoxide dismutase but not by catalase.

A colorimetric assay to quantify dehydrogenase activity in crude cell lysates.

Nitroblue tetrazolium (NBT) in the presence of phenazine methosulfate (PMS) reacts with the NADPH produced by dehydrogenases to produce an insoluble blue-purple formazan. Endpoint assays taking advantage of this reaction have been successfully used to detect the activity of several dehydrogenases. Here we present a version of this assay suitable for determining the kinetics of 6-phosphogluconate dehydrogenase catalysis in crude lysates of bacterial cells prepared in 96-well plates. Using the assay to screen a small library of variant 6-phosphogluconate dehydrogenases generated by error-prone polymerase chain reaction, we were able to identify three variants with improved activity and thermostability over the parent enzyme. These enzymes were partially purified and shown to be expressed at higher levels than the parent (leading to the increase in activity), and all three variants were indeed more thermostable than the parent (temperature midpoints 4-7 degrees C higher) after purification. Thus the NBT-PMS assay appears suitable for screening libraries of variant dehydrogenases.

The effect of apomorphine on exocytosis and metabolic burst of polymorphonuclear leukocytes.

1 In rabbit polymorphonuclear leukocytes (PMNLs) apomorphine at 10-100 microM inhibits fMet-Leu-Phe and A23187-induced exocytosis, and the phorbol myristate acetate- and fMet-Leu-Phe-induced activation of the metabolic burst. The secretory response was not restored by washing the cells after pretreatment with apomorphine. 2 The inhibitory effect of apomorphine was not prevented by the dopamine receptor antagonists haloperidol and pimozide, nor did dopamine itself inhibit fMet-Leu-Phe-induced exocytosis. It therefore seems unlikely that effects are mediated via dopamine receptors. However, sulphydryl reagents reduced the inhibitory effect of apomorphine, suggesting that it may depend upon interaction with susceptible sulphydryl groups, the intactness of which is required for exocytosis and other functions of PMNLs.

Noradrenergic-nitrergic interactions in the rat anococcygeus muscle: evidence for postjunctional modulation by nitric oxide.

1. The distribution of NADPH-diaphorase positive and catecholamine-containing nerve structures, and functional noradrenergic-nitrergic interactions, were studied in the rat anococcygeus muscle. 2. The morphological findings demonstrated NADPH-diaphorase positive neurons mostly as aggregates in intramural ganglia, nerve tracts and few single nerve fibres forming plexus-like structures. 3. The nitric oxide synthase inhibitor NG-nitro-L-arginine (L-NOARG) inhibited concentration-dependently the nitrergic relaxation, an effect reversed by L-arginine. The drug had dual effects on noradrenergic contractile responses: at lower concentrations (0.1-10 microM) it decreased the amplitude of contractions and this was not affected by L-arginine; higher concentrations (50-500 microM) potentiated the contractions, an effect that was prevented by L-arginine. 4. The electron acceptor, nitro blue tetrazolium (NBT) produced a rapid inhibition of the noradrenergic contractile responses (EC50 0.178 +/- 0.041 microM). The drug decreased the tone of the preparations. However, it potentiated concentration-dependently the nitrergic relaxations. 5. NBT (1 microM) had no significant effect on the relaxations induced by exogenously applied nitric oxide (NO)-donor sodium nitroprusside (SNP, 0.01-50 microM). However, the effect of NBT (0.1-10 microM) on the electrically induced relaxation was significantly decreased by L-NOARG (10 and 50 microM). The inhibition was of a non-competitive type. 6. Neither L-NOARG (100 microM) nor NBT (1 microM) had any effect on the spontaneous or electrically-induced release of 3H-radioactivity from the tissues preincubated in [3H]-noradrenaline. 7. It is concluded that L-arginine-NO pathway can modulate noradrenergic transmission in the rat anococcygeus muscle at postjunctional, but not prejunctional site(s).

Nitroblue tetrazolium blocks BK channels in cerebrovascular smooth muscle cell membranes.

The effects of p-nitroblue tetrazolium (NBT) on large conductance, calcium-activated potassium channels (BK channels) in enzymatically dispersed rat cerebrovascular smooth muscle cells (CVSMCs) were examined. Patch clamp methods were employed to record single BK channel currents from inside-out patches of CVMC membrane maintained at 21 - 23 degrees C. When applied to the cytoplasmic face of inside-out membrane patches (internally applied NBT), micromolar concentrations of NBT reversible reduced the mean open time of BK channels, without changing channel conductance. NBT altered the frequency distribution of BK channel open times from a two exponential to a single exponential form. In the absence of NBT, mean channel open time increased on membrane depolarization. In the presence of internally applied NBT, mean channel open became essentially independent of membrane potential. Internally applied NBT also reduced the mean closed time of BK channels when measured at membrane potentials in the range -80 mV to +20 mV. The combined effects of internal NBT on mean open and closed times resulted in the suppression of BK channel open probability when measured at positive membrane potentials. When applied to the external membrane face, micromolar concentrations of NBT reduced mean channel open time progressively as the membrane was hyperpolarized, and also reduced open probability at negative membrane potentials. A model is proposed in which NBT alters channel gating by binding to a site at or near to the cytoplasmic membrane face. Externally applied NBT suppressed BK channel open probability at concentrations which also inhibit nitric oxide synthase (NOS). Therefore, the potential role of potassium channel block in NBT actions previously attributed to NOS inhibition is discussed.