N-acetylcysteine depresses contractile function and inhibits fatigue of diaphragm in vitro

We have previously shown that antioxidant enzymes (superoxide dismutase and catalase) depress contractility of unfatigued diaphragm fiber bundles and inhibit development of acute fatigue. In the present study, we tested for similar effects of N-acetyl-cysteine (NAC), a nonspecific antioxidant approved for clinical use. Diaphragms were excised from deeply anesthetized rats. Fiber bundles were removed, mounted isometrically at 37 degrees C, and stimulated directly using supramaximal current intensity. Studies of unfatigued muscle showed that 10 mM NAC reduced peak twitch stress (P < 0.0001), shortened time to peak twitch stress (P < 0.002), and shifted the stress-frequency curve down and to the right (P < 0.05). Fiber bundles incubated in 0.1-10 mM NAC exhibited a dose-dependent decrease in relative stresses developed during 30-Hz contraction (P < 0.0001) with no change in maximal tetanic (200 Hz) stress. NAC (10 mM) also inhibited acute fatigue. Throughout 10 min of intermittent contraction at 30-40 Hz, treated bundles developed higher stresses than time-matched control bundles (P < 0.0001). NAC concentrations > or = 30 mM were toxic, causing a prompt irreversible decrease in maximal tetanic stress (P < 0.0001). Because NAC effects mimic the effects of other antioxidant agents with different mechanisms of action, we conclude that exogenous antioxidants exert stereotypical effects on contractile function that differ between unfatigued and fatiguing muscle. Unlike antioxidant enzymes, however, NAC has been approved for clinical use and may be used in future studies of human muscle fatigue.     

Experimental evidence for fatigue during sliding wear

Metallurgical and Materials Transactions A -     

Paired phrenic nerve stimuli for the detection of diaphragm fatigue in humans

The transdiaphragmatic pressure (Pdi) elicited by paired bilateral phrenic nerve stimulation may be viewed as the sum of the Pdi values produced by the first (t1) and second (t2) stimulus. The Pdi at t2 (P[di,t2]) is a function of the interstimulus interval. A reduction in the ratio obtained by dividing Pdi,t2 at 10 Hz (P[di,t2,10]) by Pdi at 100 Hz (P[di,t2,100]) (t2(10:100)) has been proposed as a test of low frequency diaphragm fatigue. The aim of the present study was to establish whether this change could also be detected using paired cervical magnetic nerve stimulation (pCMS), and whether t2(10:100) was influenced by lung volume. We studied healthy subjects at functional residual capacity (FRC), at 0.5 and 1.0 L below FRC, and at 0.5, 1.0 and 1.5 L above FRC. The subjects were then subjected to a fatiguing protocol (2 min of maximal isocapnic ventilation (MIV)). Studies were repeated at FRC 20 and 60 min after MIV and between these times at 1.0 L below and 1.5 L above FRC. In the unfatigued state, t2(10:100) had a negative relationship with increasing lung volume (r2=0.98, p=0.002). After MIV there was a fall in the Pdi elicited by a single stimulus (mean fall at 20 min 17.9% and at 60 min 14.6%, p<0.03 for both). t2(10:100) fell by a mean 28.1% after 20 min and mean 22.9% at 60 min (p<0.03 for both). This change was mainly mediated by a fall in the P[di,t2,10]. The t2(10:100) was not able to distinguish between fatigue and acute hyperinflation. We conclude that paired cervical magnetic nerve stimulation may be used to detect low frequency diaphragm fatigue but that it remains important to control for lung volume.     

Evidence of hydroxyl free radical generation by calcium overload in rat myocardium

Although calcium (Ca2+) is important in cardiac dysfunction and has also been reported as a source of oxidative toxicity, the connection between Ca2+ overload and oxygen free radicals in the myocardium is not clear. We have investigated whether Ca2+ overload generates hydroxyl free radicals in rat ventricle. HPLC with electrochemical detection was used to measure the levels of 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formed when the hydroxyl free radical reacts with salicylate. Ringer's solution containing salicylic acid (0.5 nmol microL-1 min-1) was infused through a microdialysis probe in the region of the left anterior descending coronary artery of the rat ventricle. A positive linear correlation was obtained between Ca2+ and hydroxyl free radical formation trapped as 2,3-DHBA (r2 = 0.976) and 2,5-DHBA (r2 = 0.982) in the myocardial dialysate. The administration of ouabain (1 mg kg-1, i.v.), a Ca2+ elevator, into the femoral vein significantly increased the level of 2,3- and 2,5-DHBA. These results indicate that Ca2+ overload generates hydroxyl free radicals in rat heart.     

The origin of the hydroxyl radical oxygen in the Fenton reaction

There is an ongoing discussion in the chemical literature regarding the nature of the highly reactive hydroxyl radical formed from the reaction between ferrous iron and hydrogen peroxide (the Fenton reaction). However, the fundamental experiment of directly determining the source of the hydroxyl radicals formed in the reaction has not yet been carried out. In this study, we have used both hydrogen peroxide and water labeled with 17O, together with ESR spin trapping, to detect the hydroxyl radicals formed in the reaction. ESR experiments were run in phosphate buffer with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, and either H2O2 or H2O labeled with 17O. The hydroxyl radical was generated by addition of Fe2+ ion to H2O2, or as a control, by photolysis of H2O2 in the ESR cavity. Observed ESR spectra were the sum of DMPO/.16OH and DMPO/.17OH radical adduct spectra. Within experimental accuracy, the percentage of 17O-labeled hydroxyl radical trapped by the DMPO was the same as in the original hydrogen peroxide, for either method of hydroxyl radical generation, indicating that the trapped hydroxyl radical was derived exclusively from hydrogen peroxide and that there was no exchange of oxygen atoms between H2O2 and solvent water. Likewise, the complementary reaction with ordinary H2O2 and 17O-labeled water also showed that none of the hydroxyl radical was derived from water. Our results do not preclude the ferryl intermediate, [Fe = O]2+ reacting with DMPO to form DMPO/.OH if the ferryl oxygen is derived from H2O2 rather than from a water ligand.     

溴邻苯三酚红氧化法检测Cu^＋和H2O2反应产生的羟自由基

建立了以Cu^ -H2O^-溴邻苯三酚红体系测定羟自由基的新方法。该方法基于Cu^ 和H2O2反应产生羟自由基;它能使溴邻苯三酚红的颜色发生变化,用分光光度计测定其ΔA值的变化,可间接测定羟自由基的产生量,通过研究,得到最佳实验条件。结果表明,该方法快速,稳定性好,操作简便,可作为一种简易的筛选抗氧化剂的方法。     

Scavenging activity of furan derivatives against hydroxyl radical generated by Fenton system

As the continuation of the work on the antioxidant properties of furan derivatives, we have studied hydroxyl radical (OH.) scavenging activity of dimethylfuran (DMF) and diphenylfuran (DPF), which are well-known as an effective scavenger of singlet oxygen, by an ESR spin trapping technique. The incubation mixture of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent with FE2+ and H202 in 50% acetonitrile aqueous solution gave 1:2:2:1 line signals characteristic of DMPO-OH spin adduct. The additions of furan derivatives to the incubation mixture decreased the intensity of the DMPO-OH spin adduct signal in a dose-dependent manner. The activities of furan derivatives were in the order of DMF > DPF > furan. The decrease in the intensity of the signals was not due to the inhibition of OH. generating system itself and the destruction of the spin adduct by furan derivatives. By comparison with the common OH. scavengers, DMF and DPF were found to scavenge OH. more effectively than dimethylsulfoxide and mannitol. These results indicate that DMF and DPF can act as a OH. scavenger as well as a singlet oxygen scavenger.     

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.     

Cardiac glycosides in the defensive secretion of chrysomelid beetles: evidence for their production by the insects

The defensive secretions of some chrysomelid beetles belonging to the genera Chrysolina, Chrysochloa, and Dlochrysa contain complex mixtures of cardenolides. The spectral data for some of these compounds suggest that they are monohydroxylated digitoxigenin derivatives linked to a pentose (such as xylose or arabinose). Evidence indicates that the beetles do not sequester these steroid glycosides from their host plants.     

Characterization of the reaction rate coefficient of DNA with the hydroxyl radical

Using agarose gel electrophoresis, we have measured the yield of single-strand breaks (SSBs) induced by 137Cs gamma irradiation in a variety of plasmid DNA substrates ranging in size from 2.7 kb to 38 kb irradiated in aerobic aqueous solution in the presence of the hydroxyl radical scavenger dimethyl sulfoxide (DMSO). Under these conditions DNA SSBs are caused mainly by the hydroxyl radical. Using the competition between DMSO and DNA for the hydroxyl radical, we have estimated the rate coefficient for the reaction of the hydroxyl radical with DNA. The results cannot be characterized by conventional steady-state competition kinetics. However, it is possible to describe the second-order rate constant for the reaction as a function of the scavenging capacity of the solution. The second-order rate constant increases with increasing scavenging capacity, rising from about 5 x 10(8) dm3 mol-1 s-1 at 10(5) s-1 to about 10(10) dm3 mol-1 s-1 at 10(10) s-1. This dependence of the second-order rate constant on the scavenging capacity appears to be more pronounced for larger plasmids.     

Mechanisms for facilitation of nitric oxide-evoked [3H]GABA release by removal of hydroxyl radical

We have investigated the mechanisms for enhancement of nitric oxide (NO)-evoked gamma-[3H]aminobutyric acid ([3H]GABA) release from mouse cerebrocortical neurons by hydroxyl radical (.OH) scavengers. .OH scavengers, such as N,N'-dimethylthiourea (DMTU), uric acid, and mannitol, dose-dependently facilitated NO-evoked [3H]GABA release evoked by NO liberated from S-nitroso-N-acetylpenicillamine. Ionomycin-evoked [3H]GABA release, which was significantly inhibited by hemoglobin and an NO synthase, N(G)-methyl-L-arginine, was also enhanced by DMTU. These results indicate that GABA release evoked by both endogenous and exogenous NO is facilitated by .OH scavengers. These enhancing actions of .OH scavengers were completely abolished by Ca2+ removal from incubation buffer and by an L-type voltage-dependent Ca2+ channel (VDCC) inhibitor, nifedipine, whereas each .OH scavenger showed no effects on [3H]GABA release in the absence of NO. Inhibitors for P/Q- and N-type VDCCs had no effects on the enhancement. NO-induced 45Ca2+ influx was also dose-dependently enhanced by .OH scavengers, although 45Ca2+ influx was not altered by .OH scavengers in the absence of NO. Nifedipine abolished this enhancement of the NO-induced 45Ca2+ influx by .OH scavengers. These results indicate that the removal of .OH by its scavengers facilitates the NO-evoked [3H]GABA release dependent on Ca2+ and that this enhancement is due to the increase in Ca2+ influx via L-type VDCCs.     

A case of rupture of the diaphragm caused by the plication for diaphragm eventration

The number of children who are homeless or in foster care has risen dramatically during the past two decades. Poverty, substance abuse, lack of education and employment, and the failure of the social "safety net" to catch all those in need of support and financial assistance are root causes of this increase. The Personal Responsibility and Work Opportunity Reconciliation Act of 1996, popularly know as the "welfare reform" act, will likely have a powerful impact on levels of child poverty in the future and place even greater numbers of children at risk for becoming homeless or entering foster care over the next decade. Recent studies provide increased understanding of the health care and educational needs of children who are homeless or in foster care.     

Differential control of immunoreactive alpha-inhibin and progesterone production by marmoset luteal cells in vitro: evidence for a paracrine action of alpha-inhibin on basal and gonadotropin-stimulated progesterone production

There is an increase in plasma concentrations of immunoreactive (ir) inhibin unaccompanied by a rise in plasma progesterone during early pregnancy in the marmoset monkey. We investigated the potential involvement of hCG and prostaglandin E2 (PGE2) in stimulating a selective increase in inhibin concentrations by measuring the production of ir-alpha-inhibin and progesterone by dispersed luteal cells cultured under serum-free conditions. After one day, hCG had no effect on progesterone production by the cells but stimulated a significant increase (p < 0.05) in alpha-inhibin production. PGE2 significantly increased progesterone production (p < 0.001) but inhibited the production of alpha-inhibin (p < 0.001). After three days of culture, output of alpha-inhibin fell to low levels and no significant effect of hCG or PGE2 was detected. Progesterone also fell with time in culture, but hCG maintained production resulting in a significant increase above control levels (p < 0.001). The addition of low density lipoproteins (LDL) to the culture medium increased progesterone production (p < 0.001) while decreasing alpha-inhibin production (p < 0.01). Immunoneutralization of endogenous alpha-inhibin resulted in a significant decrease in both basal (p < 0.05) and gonadotropin-stimulated (p < 0.05) progesterone concentrations. These results provide further evidence for differential control of progesterone and alpha-inhibin production by marmoset luteal cells and show that hCG can selectively stimulate alpha-inhibin production. In addition, alpha-inhibin may have a local paracrine action in the marmoset CL, enhancing both basal and gonadotropin-stimulated progesterone secretion.     

Transition-state selectivity for a single hydroxyl group during catalysis by cytidine deaminase

Cytidine deaminase binds transition-state analog inhibitors approximately 10(7) times more tightly than corresponding 3,4-dihydro analogs containing a proton in place of the 4-hydroxyl group. X-ray crystal structures of complexes with the two matched inhibitors differ only near a "trapped" water molecule in the complex with the 3,4-dihydro analog, where contacts are substantially less favorable than those with the hydroxyl group of the transition-state analog. The hydrogen bond between the hydroxyl group and the Glu 104 carboxylate shortens in that complex, and may become a "low-barrier" hydrogen bond, since at the same time the bond between zinc and the Cys 132 thiolate ligand lengthens. These differences must therefore account for most of the differential binding affinity related to catalysis. Moreover, the trapped water molecule retains some of the binding energy stabilizing the hydroxyl group in the transition-state analog complex. To this extent, the ratio of binding affinities for the two compounds is smaller than the true contribution of the hydroxyl group, a conclusion with significant bearing on interpreting difference free energies derived from substituent effects arising from chemical modification and/or mutagenesis.     

Enhancement of peroxynitrite-evoked acetylcholine release by hydroxyl radical scavengers from mouse cerebral cortical neurons

We investigated the effects of hydroxyl radical scavengers on peroxynitrite (OONO-)-evoked acetylcholine (ACh) release from mouse cerebral cortical neurons. N,N'-dimethylthiourea, a hydroxyl radical scavenger, dose-dependently increased OONO(-)-evoked ACh release. Other hydroxyl radical scavengers such as uric acid and mannitol, also enhanced OONO(-)-evoked ACh release, although these enhancing effects were not found in the absence of OONO-. In addition, OONO(-)-induced [45Ca2+]influx was significantly facilitated by the scavengers, whereas no effects of the scavengers on [45Ca2+]influx was observed in the absence of OONO-. These results indicate that hydroxyl radical scavengers enhance OONO(-)-evoked ACh release via the facilitation of OONO(-)-induced [45Ca2+]influx.     

Function of the diaphragm during exercise]

We evaluated the effect of global inspiratory muscle fatigue (GF) on respiratory muscle control during exercise at 30%, 60%, and 90% of maximal power output in normal subjects. Fatigue was induced by breathing against a high inspiratory resistance until exhaustion. Respiratory pressures, breathing pattern, and perceived breathlessness were measured. Induction of GF had no effect on the ventilatory parameters during mild and moderate exercise. It altered, however, ventilatory response to heavy exercise by increasing breathing frequency and minute ventilation, with minor changes in tidal volume. This was accompanied by an increase in perceived breathlessness. GF significantly increased both the tonic and phasic activities of abdominal muscles that allowed 1) the diaphragm to maintain its function while developing less pressure, 2) the same tidal volume with lesser shortening of the rib cage inspiratory muscles, and 3) relaxation of the abdominal muscles to contribute to lung inflation. The increased work performed by the abdominal muscles may, however, lead to a reduction in their strength. GF may impair exercise performance in some healthy subjects that is probably not related to excessive breathlessness or other ventilatory factors. The respiratory system is remarkably adaptable in maintaining ventilation during exercise even with impaired inspiratory muscle contractility.     

Additional evidence for the plastic blunting process of fatigue crack propagation

Current workers who deal with fatigue crack propagation mechanisms express the view that fracture surface striations are formed at the start of a tensile stroke instead of the end of a compressive stroke, as required by the older mechanism of the “plastic blunting process” (PBP). The current views are criticized and new fractographic evidence is presented in support of the PBP mechanism. The effects of a nonzero mean stress and of environment are also discussed. Variable amplitude tests are shown to be useful in dealing with questions about crack propagation kinetics.     

Adverse health effects of PM10 particles: involvement of iron in generation of hydroxyl radical

OBJECTIVES: Environmental particles < 10 microns average aerodynamic diameter (PM10) are associated with mortality, exacerbation of airways diseases, and decrement in lung function. It is hypothesised that PM10 particles, along with other pathogenic particles, generate free radicals at their surface in reactions involving iron, and that this is a factor in the pathogenicity of PM10 particles. Identification of free radical activity in PM10 and examination of the content and role of iron in this process was undertaken. METHODS: Free radical activity was detected with a supercoiled plasmid, phi X174 RF1 DNA, and measured as scission of the supercoiled DNA (mediated by free radicals) by scanning laser densitometry. The role of the hydroxyl radical was confirmed by the use of the specific scavenger mannitol, and the role of iron investigated with the iron chelator desferrioxamine-B (DSF-B). Iron released from PM10 particles at pH 7.2 and pH 4.6 (to mimic conditions on the lung surface and in macrophage phagolysosomes, respectively) was assessed spectrophotometrically with the Fe++ chelator ferrozine and the Fe+ + + chelator DSF-B. RESULTS: PM10 particles showed significant free radical activity by their ability to degrade supercoiled DNA. A substantial part of this activity was due to the generation of hydroxyl radicals, as shown by partial protection with mannitol. Similarly, DSF-B also conferred protection against the damage caused to plasmid DNA indicating the role of iron in generation of hydroxyl radicals. Negligible Fe++ was released at either pH 7.2 or pH 4.6 by contrast with Fe+ + +, which was released in substantial quantities at both pHs, although twice as much was released at pH 4.6. CONCLUSIONS: PM10 particles generate the hydroxyl radical, a highly deleterious free radical, in aqueous solution. This occurs by an iron dependent process and hydroxyl radicals could play a part in the pathogenicity of PM10 particles. Iron release was greatest at the pH of the lysosome (pH 4.6) indicating that iron may be mobilised inside macrophages after phagocytosis, leading to oxidative stress in the macrophages.     

Removals of hydrogen peroxide and hydroxyl radical by thiol-specific antioxidant protein as a possible role in vivo

Thiol-specific antioxidant protein (Protector Protein; PRP) from Saccharomyces cerevisiae was found to remove hydrogen peroxide and hydroxyl radical in the presence of dithiothreitol (DTT). Without DTT as a reducing equivalent, the antioxidant protein did not show the activities for destroying hydrogen peroxide and hydroxyl radical. N-ethylmaleimide (NEM) was observed to prevent the PRP from both removing hydrogen peroxide and protecting the cleavage of DNA. These observations suggest that the sulfhydryl of cysteine in PRP could function as a strong nucleophile to attack and destroy H2O2 and .OH.     

Effect of antithyroid drugs on hydroxyl radical formation and alpha-1-proteinase inhibitor inactivation by neutrophils: therapeutic implications

The release of proteolytic enzymes and generation of strong oxidants such as the hydroxyl radical by activated neutrophils has been proposed to play an important role in mediating toxin-induced liver injury. The antithyroid drug propylthiouracil protects against liver injury induced by many hepatotoxic agents and markedly reduces mortality in patients with alcoholic liver disease. However, the mechanism(s) by which propylthiouracil protects against liver injury is not well understood. The present studies investigate the effect of antithyroid drugs on proteolytic enzyme activity and on hydroxyl radical generation from activated neutrophils. In the presence of hydrogen peroxide and chloride, neutrophil myeloperoxidase, an enzyme from the same gene superfamily as thyroid peroxidase, generates hypochlorous acid which inactivates alpha-1-proteinase inhibitor (A1PI) present in serum. This inactivation allows neutrophil-released proteolytic enzymes to attack cells. In the present study myeloperoxidase activity was inhibited fully at therapeutic concentrations by antithyroid drugs (propylthiouracil and methimazole). Antithyroid drugs fully prevented hypochlorous acid formation, and prevented neutrophil-mediated inactivation of A1PI, with concomitant blockage of proteolytic activity. Conversely, generation of both superoxide and hydroxyl radicals by activated neutrophils was unaffected by propylthiouracil. The production of these oxygen radicals was fully inhibited by the NADPH oxidase inhibitor diphenylene iodonium chloride, however. These studies indicate that antithyroid drugs are unlikely to prevent cell injury by inhibiting hydroxyl radical generation or by scavenging hydroxyl radicals, but are likely to exert their hepatoprotective anti-inflammatory action by inhibiting neutrophil myeloperoxidase, an enzyme akin to thyroid peroxidase.