Diabetic-induced endothelial dysfunction in rat aorta: role of hydroxyl radicals

We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-beta signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-beta superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-beta signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-beta signals exert competing positive and negative influences on the development of muscle pioneer cells.     

Apparent role of hydroxyl radicals in oxidative brain injury induced by sodium nitroprusside

Sodium nitroprusside (disodium nitroferricyanide) has been suggested to cause cytotoxicity through either the release of cyanide and/or nitric oxide. The present study investigated a possible mechanism that after a brief release of nitric oxide, iron moiety of breakdown products of sodium nitroprusside could cause a long lasting oxidative stress, such as hydroxyl radical generation, lipid peroxidation and cytotoxicity. Intranigral administration of sodium nitroprusside (0-16.8 nmol) to rats induced an acute increase in lipid peroxidation in the substantia nigra and a chronic dopamine depletion in the caudate nucleus. Photodegraded (nitric oxide-exhausted) sodium nitroprusside, however, still produced lipid peroxidation and neurotoxicity in the midbrain. Moreover, non-iron containing nitric oxide-donor compounds, such as S-nitroso-N-acetylpenicillamine, did not cause oxidative brain injury in vivo suggesting that nitric oxide may not mediate neurotoxicity induced by sodium nitroprusside. Additional in vitro studies demonstrated that both freshly prepared (nitric oxide donor) and photodegraded (nitric oxide-exhausted) sodium nitroprusside generated hydroxyl radicals in the presence of ascorbate and also increased lipid peroxidation in brain homogenates. These pro-oxidative effects of sodium nitroprusside were blocked by nitric oxide, S-nitroso-N-acetylpenicillamine, oxyhemoglobin, and deferoxamine (iron chelator). The present results suggest that iron moiety, rather than nitric oxide, may mediate the pro-oxidative properties of sodium nitroprusside. With this new information in mind, the misuse of sodium nitroprusside as a selective nitric oxide donor in both basic and clinical uses should be urgently addressed.     

Electronic spin resonance detection of superoxide and hydroxyl radicals during the reductive metabolism of drugs by rat brain preparations and isolated cerebral microvessels

A spin trapping technique was used to analyze by electron spin resonance (ESR) the formation of oxygen-derived free radicals during the cerebral reductive metabolism of xenobiotics able to undergo a single electron reduction, i.e. quinones, pyridinium compounds and nitroheterocyclics. Paraquat, menadione and nitrofurazone were used as model compounds of these three classes of molecules. ESR spectra indicative of superoxide and hydroxyl radical formation were obtained by incubation of brain homogenates directly within the ESR cavity at 37 degrees C for each of the three molecules tested. These signals were dependent on nucleotide cofactors, and increased in a time-dependent manner. The NADPH and NADH dependent free radical production was further characterized in brain microsomal and mitochondrial fractions, respectively. By using various combinations of reactive species inactivating enzymes (superoxide dismutase, catalase), a metal chelator (deferoxamine), and an hydroxyl trapping agent (dimethylsulfoxide), it was shown that (1) the primary radical generated was the superoxide anion; and (2) a significant production of the hydroxyl radical also occurred, that was secondary to the superoxide anion production. Consistent signals indicative of the production of both oxygen-derived free radicals were obtained when isolated cerebral microvessels which constitute the blood-brain barrier were incubated with the model molecules. This is of particular toxicological relevance, because this barrier represents a key element in the protection of the brain, and is in close contact with blood-born exogenous molecules.     

The permeability transition pore opening in intact mitochondria and submitochondrial particles

Caenorhabditis elegans BM-40 (positions 19-264) and its extracellular calcium-binding domain (positions 139-264) were obtained in recombinant form from human kidney cells using an episomal expression vector. The purified proteins showed single bands of 33 kDa [BM-40-(19-264)-peptide] or 14 kDa [BM-40-(139-264)-peptide] on electrophoresis, contained internal disulfide bonds and a helices and were relatively resistant to matrix metalloproteinases. Hexosamine analysis indicated substitution by one N-linked and two O-linked oligosaccharides and recombinant BM-40 was indistinguishable in its immunological epitopes from nematode tissue-derived BM-40, suggesting that it was obtained in native form. Both recombinant C. elegans proteins showed a distinct binding activity for human collagens I and IV in solid-phase and surface-plasmon-resonance assays with an affinity (Kd = 1-2 microM), comparable to that of mammalian BM-40. However, calcium-binding studies revealed only a low-affinity site (Kd = 6.2 mM) and failed to show the characteristic conformational change upon addition of EDTA. These and a few other differences are apparently due to two extra disulfide bonds and two deletions/insertions in C. elegans BM-40 and can be partly interpreted from the X-ray structure of a large part of human BM-40. The immunological assays available and the predictions of the location of the collagen-binding epitope should facilitate a molecular and genetic approach to understand the function of BM-40 in the development of C. elegans.     

Indole-3-propionate: a potent hydroxyl radical scavenger in rat brain

The hydroxyl radical scavenging activity of indole-3-propionate was evaluated by kinetic competition studies with the hydroxyl radical trapping reagent 2,2'-azino-bis-(3-ethyl-benz-thiazoline-6-sulfonic acid) (ABTS) and by measuring hydroxyl radical-initiated lipid peroxidation in the rat striatum. Using ABTS, the indole was shown to act as a potent hydroxyl radical scavenger with a rate constant of 7.8x1010 mol l-1 s-1. Hydroxyl radical-initiated lipid peroxidation, determined by measuring tissue malondialdehyde formation, was inhibited dose-dependently both in vitro and in vivo. Indole-3-propionate reacts with hydroxyl radicals at a diffusion controlled rate and can thereby provide on-site protection against the oxidative damage of biomolecules induced by these highly reactive and toxic oxygen intermediates. While it remains to be established if endogenous brain tissue levels of indole-3-propionate are sufficiently high to have a significant impact on total antioxidative capacity, the compound itself or a structurally related agent may be useful as an antioxidant adjuvant to combat hydroxyl radical-mediated oxidative stress.     

Production of hydroxyl radicals in contracting skeletal muscle of cats

Reactive oxygen species increase during exhaustive contraction of skeletal muscle, but characterization of the specific species involved and their rates of production during nonexhaustive muscle contraction have not been investigated. We hypothesized that the production rate of hydroxyl radical (.OH) increases in contracting muscle and that this rate is attenuated by pretreatment with deferoxamine (Def) or dimethylthiourea (DMTU). We measured the rate of production of .OH before, during, and after 5 min of intermittent static contraction of the triceps surae muscles in cats (n = 6) using the formation of p-, m-, and o-tyrosines by hydroxylation of phenylalanine. L-Phenylalanine (30 mg/kg i.v.) was administered to each animal 3 min before contraction. Blood samples were collected from the popliteal vein 1 min before contraction; 1, 3, and 4.5 min during contraction; and 1 min after contraction. During and after contraction, the cumulative production rates of p-, m-, and o-tyrosines were elevated by 42.84 +/- 5.41, 0.25 +/- 0.04, and 0.21 +/- 0.03 nmol.min-1.g-1, respectively, compared with noncontracting triceps surae muscles. Pretreatment with Def (10 mg/kg i.v.; n = 5) or DMTU (10 mg/kg i.v.; n = 4) decreased the cumulative rates of production of p-, m-, and o-tyrosines during and after contraction. Additionally, the rate of tyrosine production increased in proportion to the percentage of maximal tension developed by the triceps surae muscles. These results directly demonstrate that .OH is produced in vivo in the skeletal muscle of cats during intermittent static contraction and that production can occur before the onset of fatigue.     

Reactivity of aliphatic peptides toward hydroxyl radicals in aqueous solution

Dextrostix is now widely used as a method of screening for hypoglycemia of the newborn. There has always been some anxiety about its accuracy for estimating very low blood sugars; this is important, since symptomatic hypoglycemia of the newborn does not usually occur until the blood glucose falls below 20 mg/dl. In 1970 a reflectance meter was introduced which would measure the colour of the strip electronically. The aim of this study was to assess the accuracy of the meter and its reliability in estimating hypoglycemia. The study was continued to investigate a new model of the reflectance meter which superseded the original one. In the pilot study using the original model, blood samples were taken from 46 babies. Readings of the Dextrostix were made by two independent observers and compared with a reading taken on the reflectance meter. These estimations were later compared with blood glucose measured by the glucose oxidase method. the meter showed a marked tendency to overestimate the blood glucose: 44 out of 46 samples were overestimated (Fig. 1, 2). In the second series 180 cord blood samples were collected. Because the intention was to study very low levels of blood glucose, the samples were allowed to stand at room temperature for several hours to allow glycolysis to occur. Again, readings were taken by two independent observers and compared with the readings taken on the new Dextrostix-Eyetone meter. The blood glucose was measured on each of the samples. There was a strong correlation (r = 0.8877, p less than 0.00005) between the blood glucose values and the readings taken from the meter, with no tendency towards overestimation (Fig. 3). A similar correlation (r = 0.8533, p less than 0.00005) was seen for the observers' readings and the chemical method, although there was a tendency to underestimate blood glucose (Fig. 4). When the meter gave an estimate of more than 20 ml/dl, in no case was the actual blood glucose in the profoundly hypoglycemic group of less than 10 mg/dl. When estimated by eye there was one case in which the blood glucose was only 7 mg/dl but the observer had estimated the result of 30 mg/dl. In order to avoid any possibility that a blood glucose was less than 20 mg/dl, it is necessary to take action on any estimate below 40 mg/dl on a Dextrostix. Despite this, Dextrostix remains a very useful method of screening for neonatal hypoglycemia, whether assessed by eye or with the new Dextrostix-Eyetone meter. the meter does give better results than estimating Dextrostix by eye, but the difference was never statistically significant.     

Measurement of the membrane potential generated by complex I in submitochondrial particles

To investigate the energy-conserving function of the NADH:ubiquinone reductase (complex I), we have selected oxonol VI [bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol] as the most sensitive probe for measuring the reactions of membrane potential generation in submitochondrial particles. Calibration of the oxonol signals with potassium diffusion potentials shows a non-linear response after a threshold around -50 mV. Thermodynamic evaluations indicate that the upper limit of the oxonol response to the potential generated by complex I is around -220 mV, which is close to the maximal protonmotive force in coupled submitochondrial particles. NADH addition to particles in which ubiquinol oxidation is blocked by inhibitors of other respiratory complexes generates oxonol signals corresponding to membrane potentials of -130 to -180 mV. These signals are produced by about four turnovers of the complex reducing endogenous ubiquinone (i.e. non-steady-state conditions) and are equivalent to a charge separation similar to that of the antimycin-sensitive reactions of ubiquinol:cytochrome c reductase (complex III). The transient oxonol signals under non-steady-state conditions are thus informative of crucial steps in the electrogenic reactions catalyzed by complex I. The possible nature of these electrogenic reactions is discussed in relation to proposed mechanisms for complex I.     

Effects of hydroxyl radicals on KATP channels in guinea-pig ventricular myocytes

We studied the effects of oxygen free radicals on the ATP-sensitive potassium channel (KATP channel) of guinea-pig ventricular myocytes. Single KATP channel currents were recorded from inside-out patches in the presence of symmetrical K+ concentrations (140 mM in both bath and pipette solutions). Reaction of xanthine oxidase (0.1 U/ml) on hypoxanthine (0.5 mM) produced superoxide anions (.O2-) and hydrogen peroxide (H2O2). Exposure of the patch membrane to.O2- and H2O2 increased the opening of KATP channels, but this activation was prevented by adding 1 microM glibenclamide to the bath solution. In the presence of ferric iron (Fe3+: 0.1 mM), the same procedure produced hydroxyl radicals (.OH) via the iron-catalysed Haber-Weiss reaction.OH also activated KATP channels; however, this activation could not be prevented by, even very high concentrations of glibenclamide (10 microM). These different effects of glibenclamide suggest that the mode of action of these oxygen free radicals on KATP channels is different and that.OH is more potent than.O2-/H2O2 in activating KATP channels in the heart.     

Effects of hydroxyl radicals on purified angiotensin I converting enzyme

Somatic angiotensin-converting enzyme (ACE) is a protein which contains two similar domains (N and C), each possessing a functional active site. The relationship between ACE, its natural substrates and oxygen free radicals is starting to be explored. On one hand, superoxide anions production is induced by angiotensin II and on the other hand, activated polynuclear neutrophils, through free radicals generation, alter endothelial ACE activity. In this study, we examined the impact of hydroxyl radicals (.OH) on purified ACE. .OH were produced using a generator: 2,2'-azo-bis 2-amidinopropane (GRH) provided by Lara-Spiral (Fr). GRH (3 mM), in a time-dependent fashion, inhibited ACE activity. When ACE was co-incubated for 4 h with GRH, its activity decreased by 70%. Addition of dimethylthiourea (DMTU: 0.03 to 1 mM) or mannitol + methionine (20/10 mM), two sets of .OH scavengers, produced a dose-dependent protection on ACE activity. To examine whether oxidation of thiol groups in the ACE molecule could be involved in the action of GRH, the effects of thiol reducing agents: mercaptoethanol and dithiotreitol (DTT) were investigated. These compounds produced a dose-dependent and significant protection; with 100% protection at 0.2 and 0.3 mM for mercaptoethanol and at 0.1 mM for DTT. The hydrolysis of two natural and domain-specific substrates were also explored. The hydrolysis of angiotensin I preferentially cleaved by the C domain was significantly (p < 0.01) inhibited by 57, 58 and 69% in contact with 0.3, 1 and 3 mM GRH [in nmol angio II formed/min/nmol of ACE, n = 4; 35.9 +/- 0.6 (control), 15.5 +/- 2.8 (GRH : 0.3 mM), 15.1 +/- 0.5 (1), 10.9 +/- 0.6 (3)]. The hydrolysis of the hemoregulatory peptide (hp), preferential substrate for the N domain was not affected by GRH at 0.3 mM and inhibited by 28% (not significant) by 1 mM GRH [in nmol ph hydrolized/min/nmol ACE, n = 4; 12.6 +/- 1.9 (control), 14.9 (GRH : 0.3 mM), 8.3 +/- 4.0 (1). These results demonstrated that .OH affect ACE activity and could suggest a privileged impact of GRH on the C domain. The precise sites of action of .OH remain unknown. The Cys residues near the active centers, by forming disulphide bridges during the oxidation could be of critical importance. Further studies will be needed to determine whether oxidative stress again ACE can be involved in the genesis of inflammatory vascular pathologies.     

Optical trapping and manipulation of neutral particles using lasers

The techniques of optical trapping and manipulation of neutral particles by lasers provide unique means to control the dynamics of small particles. These new experimental methods have played a revolutionary role in areas of the physical and biological sciences. This paper reviews the early developments in the field leading to the demonstration of cooling and trapping of neutral atoms in atomic physics and to the first use of optical tweezers traps in biology. Some further major achievements of these rapidly developing methods also are considered.     

Effects of cytisine on hydroxyl radicals in vitro and MPTP-induced dopamine depletion in vivo

Energy content of feedstuffs has to be determined by animal experiments. For practical purposes energy content of feeds was predicted by data on in vivo digestibility of nutrients, in vitro digestibility and different chemical parameters. Especially chemical and in vitro parameters were used in different combinations as predictors. The accuracy of such equations has to be discussed as well as cost and feasibility.     

Involvement of hydroxyl radicals in neurogenic airway plasma exudation and bronchoconstriction in guinea-pigs in vivo

1. Cigarette smoke induces plasma exudation in the airways of rodents by activation of capsaicin-sensitive 'sensory-efferent' nerves. The response is mediated predominantly by substance P (SP) and the magnitude of exudation is regulated by neutral endopeptidase (NEP). The component(s) of the smoke responsible for the activation of the nerves may be reactive oxygen radicals. We investigated the effect of the hydroxyl radical scavenger dimethylthiourea (DMTU), a regulator of superoxide anion, superoxide dismutase (SOD), and a regulator of hydrogen peroxide, catalase, on plasma exudation (measured using Evans blue dye) induced by cigarette smoke in guinea-pig main bronchi in vivo. The effect of DMTU on plasma exudation and non-cholinergic bronchoconstriction (measured as pulmonary insufflation pressure, PIP) induced by electrical stimulation of the vagus nerves was also assessed. Interaction between hydroxyl radicals and NEP was assessed with the NEP inhibitor phosphoramidon. 2. In each of the experiments, cigarette smoke increased plasma exudation by approximately 200% above air-exposed controls. Acute administration of DMTU (1.5 g kg-1, i.v. for 20 min) significantly reduced cigarette smoke-induced plasma exudation by 69%. In contrast, neither SOD (240,000 u kg-1, i.v.) nor catalase (400,000 u kg-1, i.v.) significantly affected the exudative response. 3. Chronic pretreatment with DMTU (1.25 g kg-1 over 4 days) significantly reduced bronchial plasma exudation induced by cigarette smoke by 72%. Phosphoramidon (1.5 mg kg-1, i.v.) completely reversed the inhibition by DMTU of cigarette smoke-induced plasma exudation. 4. Vagal stimulation increased plasma exudation by approximately 200% and PIP by approximately 250%. Acute treatment with DMTU had no significant inhibitory effect on these responses, whereas chronic pretreatment inhibited them by approximately 80%. Phosphoramidon reversed the inhibition by chronic DMTU. 5. SP (1 nmol kg-1) increased plasma exudation by approximately 250%, a response which was not inhibited by either acute or chronic DMTU. 6. We conclude that hydroxyl radicals, rather than superoxide anion or hydrogen peroxide, are involved in the induction of neurogenic plasma exudation and bronchoconstriction induced by cigarette smoke or by electrical stimulation of the vagus nerves. These radicals also affect the activity of NEP. Acute DMTU may affect directly the neural actions of hydroxyl radicals contained in the cigarette smoke. Chronic pretreatment with DMTU may inhibit the neurogenic airway responses by effects on tachykinin biosynthesis and/or axonal transport.     

Quantitative analysis on hydrogen trapping of TiC particles in steel

The change in the hydrogen-trapping behavior of a TiC particle accompanying its coherent to incoherent interfacial-character transition in a 0.05C-0.20Ti-2.0Ni steel that was quenched and tempered in a partially protective argon atmosphere and in ultrahigh vacuum (UHV) has been studied by thermal desorption spectrometry (TDS). The results indicated that (semi)coherent TiC precipitates demonstrate distinctly different hydrogen-trapping features from that of incoherent TiC particles with respect to hydrogen capacity, interaction energy with hydrogen, locations available for hydrogen occupation, and the capability of hydrogen absorption from the environment. The broad (semi)coherent interface of the disc-shaped (semi)coherent TiC precipitate does not trap hydrogen during tempering in a partially protected argon atmosphere, but traps hydrogen during cathodic charging at room temperature. The semicoherent interface traps 1.3 atoms/nm² of hydrogen at the core of the misfit dislocation with short-time charging (1 hour), which is characterized by a desorption activation energy of 55.8 kJ/mol. The side interface of the (semi)coherent TiC precipitate acts like the broad interface when the precipitate is small. As the precipitate grows, the side interface gradually loses its coherency and results in a simultaneous increase in the trapping activation energy and the binding energy. An increase in the trapping activation energy, i.e., the energy barrier for trapping, makes hydrogen trapping more difficult in cathodic charging at room temperature, while an increase in the binding energy enhances the capability of hydrogen absorption from the atmosphere during heat treatment. An incoherent TiC particle is not able to trap hydrogen during cathodic charging at room temperature due to its high energy barrier for trapping, but absorbs hydrogen during heat treatment at high temperatures. The amount of hydrogen that is trapped by incoherent TiC particles depends on their volume, which strongly indicates that incoherent TiC particles trap hydrogen within them rather than at the particle/matrix interface. Octahedral carbon vacancies are supposedly the hydrogen trap sites in incoherent TiC particles.     

Protonophoric activity of NADH coenzyme Q reductase and ATP synthase in coupled submitochondrial particles from horse platelets

A method to prepare coupled submitochondrial particles from horse platelets is described. The method allowed us to study the protonophoric activities of both complex I and complex V following the fluorescence quenching of the monoamine 9-amino-6-chloro-2 methoxyacridine (ACMA), a probe highly sensitive to the generation of a transmembrane delta pH. We carried out a kinetic analysis of each enzyme complex studying the proton translocation and the electron transfer activities of complex I as well as the proton translocation and the ATP hydrolytic activities of complex V. A micromethod to prepare coupled submitochondrial particles from platelets might be useful to investigate cell bioenergetic damage occurring in mitochondrial diseases and ageing.