Oxidation of tryptophan by redox intermediates of myeloperoxidase and inhibition of hypochlorous acid production

Abstract

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (k_I 2.1×10⁶ M^-1s^-1), it reacts sluggishly with compound II (k_II 7 M^-1s^-1). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I₅₀) at a concentration of 2 µM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I₅₀ 80 µM) unless superoxide dismutase was present (I₅₀ 5 µM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.     

Growth,Metabolite Profile,Oxidative Status,and Phytohormone Levels in the Green Alga <Emphasis Type="Italic">Acutodesmus obliquus</Emphasis> Exposed to Exogenous Auxins and Cytokinins

The effects of auxins and cytokinins at the range of concentrations 0.0001–100 µM on Acutodesmus obliquus (Chlorophyceae) cultures were studied. Microalga exhibited sensitivity to cytokinins in the following order: 0.01 µM tZ?>?0.1 µM Kin?>?1 µM DPU, whereas the hierarchy of auxin activity was: 0.01 µM IAA?>?0.1 µM IBA?>?0.1 µM PAA. Cytokinins possessed higher stimulating properties on the cell number, whereas auxins increased the size of cells. Differences in the metabolite profiles of the cultures treated with phytohormones were observed. Auxins and cytokinins had a positive effect on the photosynthetic apparatus enhancing the level of chlorophylls, carotenes, and xanthophylls. In comparison with auxins, cytokinins more effectively delayed oxidative damage by increasing the level of non-enzymatic antioxidants (ascorbate, glutathione) and the activity of enzymes scavenging reactive oxidative species (catalase, glutathione reductase, ascorbate peroxidase). On the other hand, auxins stimulated superoxide dismutase activity and provoked hydrogen peroxide generation, which may be involved in cell enlargement. All phytohormones reduced the content of abscisic acid and controlled the level of endogenous auxin and cytokinins suggesting complex interactions. Different dynamics of A. obliquus responses to auxins and cytokinins clearly demonstrated their diverse roles in algal growth and metabolism.     

Inhibition by BN 52021 (ginkgolide B) of the binding of [3H]-platelet-activating factor to human neutrophil granulocytes

The inhibitory effect of BN 52021, a specific antagonist of platelet-activating factor (PAF) on PAF-induced activation of human polymorphonuclear granulocytes (PMNL) and on the binding of [3H]-PAF to neutrophils were examined. BN 52021 over the range of 10(-9)-10(-4) M inhibited PAF-induced degranulation and superoxide production of PMNLs in a dose-dependent manner with Kd values of 0.6 +/- 0.1 x 10(-6) M and 0.4 +/- 0.1 x 10(-6) M, respectively. BN 52021 (up to 1 mM) did not show any agonistic activity and it did not affect neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine or leukotriene B4. The Ki value of BN 52021 for the specific binding of [3H]-PAF to neutrophils was 1.3 +/- 0.5 x 10(-6) M versus a Ki of 1.1 +/- 0.3 x 10(-7) M for PAF itself. BN 52021 did not affect metabolism of PAF by PMNL. These studies indicate that BN 52021 inhibits neutrophil responses to PAF by inhibiting binding of PAF to its specific PMNL receptor.     

Effect of exogenous nitric oxide on sulfur and nitrate assimilation pathway enzymes in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) under drought stress

The present study aimed at investigating the effects of foliar applied nitric oxide (as SNP [sodium nitroprusside]) on sulfur (glutathione reductase, guaiacol peroxidase, and glutathione S-transferase) and nitrate assimilation (nitrite and nitrate reductase) pathway enzymes in maize (Zea mays L.) exposed to water deficit conditions. The seedlings of a drought tolerant (NK8711) and sensitive (P1574) maize hybrid were applied with various SNP doses (0, 50, 100, 150, and 200 µM) under normal and drought stress conditions. Foliar spray of 100 µM markedly improved water status and chlorophyll contents and alleviated drought-induced oxidative damages through increased antioxidant (catalase, ascorbate peroxidase, and superoxide dismutase) activities in both maize hybrids. Moreover, exogenous SNP supply increased nitrite and nitrate reductase activities and upregulated glutathione reductase, glutathione S-transferase, and guaiacol peroxidase compared to no SNP supply. Interestingly, the negative effects of excess NO generation at high SNP doses (150, 200 µM) were more pronounced in P1574 than NK8711 leading to lower biomass accumulation in drought-sensitive hybrid.     

Nickel-induced oxidative stress and the role of antioxidant defence in rice seedlings

Seedlings of rice (Oryza sativa L.) cv. Pant-12 grown in sand cultures containing 200 and 400 μM NiSO₄, showed a decrease in length and fresh weight of roots and shoots. Nickel was readily taken up by rice seedlings and the concentration was higher in roots than shoots. Nickel-treated seedlings showed increased rates of superoxide anion (O₂ ^•− ) production, elevated levels of H₂O₂ and thiobarbituric acid reactive substances (TBARS) demonstrating enhanced lipid peroxidation, and a decline in protein thiol levels indicative of increased protein oxidation compared to controls. With progressively higher Ni concentrations, non-protein thiol and ascorbate (AsA) increased, whereas the level of low-molecular-weight thiols (such as glutathione and hydroxyl-methyl glutathione), the ratio of these thiols to their corresponding disulphides, and the ratio of AsA to dehydroascorbic acid declined in the seedlings. Among the antioxidant enzymes studied, the activities of all isoforms of superoxide dismutase (Cu-Zn SOD, Mn SOD and Fe SOD), guaiacol peroxidases (GPX) and ascorbate peroxidase (APX) increased in Ni-treated seedlings, while no clear alteration in catalase activity was evident. Activity of the ascorbate-glutathione cycle enzymes monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR)—significantly increased in Ni-treated seedlings. However such increase was apparently insufficient to maintain the intracellular redox balance. Results suggest that Ni induces oxidative stress in rice plants, resulting in enhanced lipid peroxidation and decline in protein thiol levels, and that (hydroxyl-methyl) glutathione and AsA in conjunction with Cu-Zn SOD, GPX and APX are involved in stress response.     

Nitric oxide improves chilling tolerance of maize by affecting apoplastic antioxidative enzymes in leaves

The effects of nitric oxide (NO) on chilling tolerance (freezing injury, ice nucleation activity, contents of hydrogen peroxide and superoxide anion, and lipid peroxidation level) and the activities of apoplastic antioxidant enzymes (peroxidase and superoxide dismutase) were investigated in the leaves of maize (Zea mays) exposed to short-term chilling. NO treatment was carried out through spraying of sodium nitroprusside (SNP), which is a donor of NO, in concentrations of 0.0, 0.1 and 1 μM on the leaves of 10-day plants. The plants then were transferred into the chilling condition (10/7 °C) 2 days before the harvesting of leaves (14th and 21th days). Application of 0.1 μM NO had more effect on the alleviation by decreasing the freezing injury in maize at least for 11 days after the application. Both concentrations of NO generally increased ice nucleation activity of apoplastic proteins extracted from leaves. The SNP applications decreased the contents of reactive oxygen species such as hydrogen peroxide and superoxide anion and the level of lipid peroxidation, while further increasing the activities of the apoplastic antioxidant enzymes studied. The results show that exogenous NO treatment provides important contributions to increasing the chilling tolerance of maize by regulating the biochemical mechanisms of chilling response, including apoplastic antioxidant enzymes. It can be seen that the NO treatment can play positive roles in alleviating chilling-induced damage in maize. Therefore, it is suggested that NO treatments may contribute to research studies related to diminishing chilling-induced damage in agricultural applications.     

Selenium improves the antioxidant ability against aluminium-induced oxidative stress in ryegrass roots

We investigated the role of selenium (Se) against aluminium (Al) stress in ryegrass by evaluating the growth responses and the antioxidant properties of plants cultured hydroponically with Al (0 or 0.2 mM) and selenite (0–10 µM Se). Al addition significantly reduced the yield and length of shoots and roots, and most Al was accumulated in the roots. Al also enhanced lipid peroxidation and activated the peroxidase (POD), ascorbate peroxidase (APX) and superoxide dismutase (SOD) enzymes in the roots. Se application up to 2 µM improved root growth and steadily decreased thiobarbituric acid reactive substances (TBARS) accumulation in plants treated with 0 and 0.2 mM Al. However, above 2 µM, Se induced stress in plants grown with or without Al. Significant changes in antioxidant enzymes activities were also found as a result of the added Se. At low Se addition levels POD was activated, whereas APX activity decreased irrespective of added Al. Furthermore, Se supplied up to 2 µM greatly decreased root SOD activity in Al-stressed plants. Our study provides evidence that Se alleviated the Al-induced oxidative stress in ryegrass roots through the enhancement of the spontaneous dismutation of superoxide radicals and the subsequent activation of POD enzyme.     

Mercury-induced changes in growth variables and antioxidative enzyme activities in Indian mustard

Abstract

In a hydroponic system, experiments were conducted to study the effect of different levels of mercury treatments (0, 5, 10, 25 and 50 µM Hg) on Indian mustard (Brassica juncea L. Czern & Coss.) cv. Pusa Jai Kisan. Concentration-dependent inhibitory effects were observed on growth characteristics (plant dry mass, leaf area, shoot and root length). These were accompanied by an increase in shoot Hg content and in oxidative stress characteristics such as the MDA and H₂O₂ levels. The plant growth decreased maximally at 50 µM of Hg. Despite a reduction in growth, activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) were enhanced with increase in Hg-treatments. The Hg-induced alterations in growth are linked with increase in lipid peroxidation (MDA and H₂O₂), whereas the enhancement in activities of antioxidant enzymes protects plants from Hg-induced oxidative stress.     

Identification of formyl peptides from Listeria monocytogenes and Staphylococcus aureus as potent chemoattractants for mouse neutrophils

The prototypic formyl peptide N-formyl-Met-Leu-Phe (fMLF) is a major chemoattractant found in Escherichia coli culture supernatants and a potent agonist at human formyl peptide receptor (FPR) 1. Consistent with this, fMLF induces bactericidal functions in human neutrophils at nanomolar concentrations. However, it is a much less potent agonist for mouse FPR (mFPR) 1 and mouse neutrophils, requiring micromolar concentrations for cell activation. To determine whether other bacteria produce more potent agonists for mFPR1, we examined formyl peptides from Listeria monocytogenes and Staphylococcus aureus for their abilities to activate mouse neutrophils. A pentapeptide (N-formyl-Met-Ile-Val-Ile-Leu (fMIVIL)) from L. monocytogenes and a tetrapeptide (N-formyl-Met-Ile-Phe-Leu (fMIFL)) from S. aureus were found to induce mouse neutrophil chemotaxis at 1-10 nM and superoxide production at 10-100 nM, similar to the potency of fMLF on human neutrophils. Using transfected cell lines expressing mFPR1 and mFPR2, which are major forms of FPRs in mouse neutrophils, we found that mFPR1 is responsible for the high potency of fMIVIL and fMIFL. In comparison, activation of mFPR2 requires micromolar concentrations of the two peptides. Genetic deletion of mfpr1 resulted in abrogation of neutrophil superoxide production and degranulation in response to fMIVIL and fMIFL, further demonstrating that mFPR1 is the primary receptor for detection of these formyl peptides. In conclusion, the formyl peptides from L. monocytogenes and S. aureus are approximately 100-fold more potent than fMLF in activating mouse neutrophils. The ability of mFPR1 to detect bacterially derived formyl peptides indicates that this important host defense mechanism is conserved in mice.     

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.     

Polymorphonuclear leucocyte (PMN) inhibitory factor prevents PMN-dependent endothelial cell injury by an anti-adhesive mechanism

Neutrophil inhibitory factor (NIF), a 41-kD glycoprotein isolated from the canine hookworm, inhibits CD11b/CD18-dependent neutrophil adhesion by binding to CD11b. We studied the effects of NIF on neutrophil-dependent endothelial cell injury using bovine pulmonary microvessel endothelial cells grown on microporous filters. Endothelial injury was determined as an increase in the transendothelial ¹²⁵I-albumin clearance rate (a measure of transendothelial permeability). Layering of neutrophils on the endothelial cell monolayer (ratio of 10 neutrophils: 1 endothelial cell) followed by activation of neutrophils with 500 nM of phorbol 12-myristate 13-acetate (PMA) increased transendothelial permeability of albumin by 3- to 4-fold over control monolayers. Pretreatment of neutrophils with NIF at concentrations of 100 nM and above prevented the increased permeability. Pretreatment of neutrophils with the anti-CD18 monoclonal antibody (mAb) IB4 similarly prevented the increase of permeability. Pretreatment of neutrophils with OKM-1, a control isotype-matched mAb directed against an irrelevant epitope on CD11b mAb, did not affect the neutrophil-dependent increase in permeability. NIF reduced the adhesion of neutrophils at concentrations of ≥100 nM and this effect was abolished by an anti-NIF polyclonal Ab. However, NIF did not prevent the generation of superoxide anions following PMA-induced activation of neutrophils layered on endothelial cell. These findings indicate that NIF inhibits the neutrophil-dependent endothelial injury by preventing CD11b/CD18-mediated neutrophil adhesion, but without altering the oxidant generating capacity of neutrophils interacting with the endothelial cell monolayer. J. Cell. Physiol. 171:212–216, 1997. © 1997 Wiley-Liss, Inc.     

Dipyridamole inhibits hydroxylamine augmented nitric oxide (NO) production by activated polymorphonuclear neutrophils through an adenosine-independent mechanism

Polymorphonuclear neutrophils (PMN) are thought to play a role in reperfusion injury and ischemia. These effects are partly mediated by toxic oxygen species (superoxide anion, hydrogen peroxide and hydroxyl radical) acting at the level of the endothelium. It was demonstrated recently that the superoxide anion reacts with nitric oxide (NO) and that interaction leads to the generation of highly toxic peroxynitrite. Several drugs were tested so far in order to affect PMN function. It was demonstrated that dipyridamole (2,6-bis-diethanolamino-4,8-dipiperidinopyrimido-(5,4-d)-pyrimidine) can influence neutrophil function by inhibiting adenosine uptake. However, this action can not fully explain all of the observed effects of dipyridamole action on PMN metabolism. The aim of our study was to evaluate the influence of dipyridamole on nitric oxide production by activated polymorphonuclear neutrophils. Incubation of PMNs with hydroxylamine (HA) and phorbol myristate acetate (PMA) generated nitrite (36.4+/-4.2 nmol/h 2x10(6) PMN), dipyridamole at 100 micromol/l, 50 micromol/l and 10 micromol/l caused a considerable drop in nitrite production (11.8+/-1.8, 19.7+/-2.7 and 27.4+/-3.2 nmol/h, respectively). Neither adenosine nor the adenosine analogue could mimic the dipyridamole effect. Moreover theophylline, an adenosine inhibitor could not reverse the dipirydamole action on PMN metabolism. We also found that dipyridamole inhibited hydrogen peroxide release from neutrophils. Catalase that scavenges hydrogen peroxide also largely abolished nitric oxide release from PMN. It is evident that dipyridamole inhibits hydroxylamine-augmented nitric oxide production by activated polymorphonuclear neutrophils through an adenosine-independent mechanism.     

Responses of in vitro-cultured <Emphasis Type="Italic">Allium hirtifolium</Emphasis> to exogenous sodium nitroprusside under PEG-imposed drought stress

Drought stress is a major threat to plant production in semi-arid and arid areas of the world. This research was laid out to asses the effects of sodium nitroprusside (SNP) as a nitric oxide donor on growth, physiological and biochemical changes of in vitro-cultured Allium hirtifolium under polyethylene glycol (PEG) induced drought stress. Basal plate explants of A. hirtifolium were cultured on MS medium containing different levels of PEG (0, 2, 4, 8 and 16 mM) and SNP (0, 10, 40 and 70 µM). After prolonged drought, growth responses, oxidative stress indicators, and phytochemical variations of regenerated plantlets with or without PEG and/or SNP treatments were recorded. Water limitation reduced regeneration potential of explants and consequently number of shoots per explant. Relative water content, total chlorophyll and carotenoid contents of regenerated A. hirtifolium plantlets decreased, but accumulation of malondialdehyde, H₂O₂ and proline and the activities of superoxide dismutase, ascorbate peroxidase, catalase and peroxidase enzymes increased with decreasing water availability. Total phenol and allicin contents were also increased in response to drought stress. Exogenous SNP in 10 and particularly in 40 µM was effective in enhancing regeneration rate and relative water content as well as protecting photosynthetic pigments under different levels of water availability. SNP also inhibited the hydrogen peroxide (H₂O₂) accumulation and lipid peroxidation in cell membranes via increasing the activities of superoxide dismutase and ascorbate peroxidase enzymes and accumulating proline and allicin. In general, these results suggest that exogenous SNP at 40 µM not only could somewhat protect A. hirtifolium from drought stress, but also can help to improve the propagation and allicin production of that plant under in vitro condition.     

Detection of phagocyte-derived free radicals with spin trapping techniques: effect of temperature and cellular metabolism

Human neutrophils activated with either particulate or soluble stimuli generate oxygen-centered free radicals which are detected by spin trapping in conjunction with electron spin resonance (ESR) spectroscopy. We investigated the effect of temperature on ESR spectra resulting from stimulation of human neutrophils with phorbol myristate acetate (PMA) or opsonized zymosan in the presence of the spin trap, 5,5-dimethyl-1-pyrroline 1-oxide (DMPO). At 20 degrees C with either stimuli, neutrophil superoxide production was manifested predominantly as the superoxide spin-trapped adduct, 5,5-dimethyl-5-hydroperoxy-1-pyrrolidinyloxy (DMPO-OOH). In contrast, at 37 degrees C, the hydroxyl spin-trapped adduct, 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH) was dominant. No evidence of hydroxyl radical (defined as the methyl spin-trapped adduct, 2,2,5-trimethyl-1-pyrrolidinyloxy, DMPO-CH3) was observed, suggesting that elevated temperatures increased the rate of DMPO-OOH conversion to DMPO-OH. In addition, the elevated temperature activated a neutrophil reductase which accelerated the rate of DMPO-OH reduction to its corresponding hydroxylamine, 2,2-dimethyl-5-hydroxy-1-hydroxypyrrolidine. This bioreduction was dependent upon the presence of both superoxide and a phagocyte-derived factor (possibly a thiol) released into the surrounding media.     

The effect of zinc on human trophoblast proliferation and oxidative stress

Adequate Zinc (Zn) intake is required to prevent multiple teratogenic effects however deviations from adequate Zn intake, including high maternal Zn status, have been linked to increased incidence of pregnancy complications, including those associated with inadequate placentation. Using placental trophoblast HTR8/SVneo cells and first trimester human placental explants (n = 12), we assessed the effects of varying Zn concentrations on trophoblast proliferation, viability, apoptosis and oxidative stress. Compared to physiologically normal Zn levels (20 µM), HTR-8/SVneo cell proliferation index was significantly lower in the presence of physiologically elevated (40 µM; P = .020) and supra-physiological (80 µM; P = .007) Zn. The latter was also associated with reduced proliferation (P = .004) and viability (P < .0001) in cultured placental explants, but not apoptosis. Reactive oxygen species production in HTR8/SVneo cultures was significantly higher in the presence of 80 µM Zn compared to all physiologically relevant levels. Oxidative stress, induced by an oxidizing agent menadione, was further exacerbated by high (80 µM) Zn. Zn did not affect lipid peroxidation in either HTR8/SVneo cells or placental explants or antioxidant defense mechanisms that included glutathione reductase and superoxide dismutase. Further study should focus on elucidating mechanisms behind impaired trophoblast proliferation and increased oxidative stress as a result of elevated Zn levels.     

The Lipid Peroxidation Product 4-Hydroxynonenal is Formed by - and is able to Attract - Rat Neutrophils in vivo

4-Hydroxynonenal (HNE), a major aidchydic product of lipid peroxidation, is a chemoattractant for neutrophilic polymorphonuclear granulocytes in vitro. The question was studied, whether HNE is formed during the ingress of neutrophils in the Sephadex model of inflammation. The polydextrane Sephadex G-200, which causes an acute aseptic traumatic inflammation, was injected subcutaneously into rats. The implants were excised 6-36 hours later, and the neutrophils separated from the exsudate by centrifugation. After extraction with dichloromethane HNE was identified in the exsudate by non-derivative reversed phase HPLC in combination with on-line uv-spectroscopy. The concentration of HNE in the inflammatory focus did not correlate with the number of neutrophils present. While the peak of HNE coincided with the time point of the highest turnover rate of neutrophils (0.13 μM at 6 hrs after implantation), the highest number of neutrophils (about 100 million cells) occurred not earlier than 18 hrs later (24 hrs after onset of inflammation).

When neutrophils were isolated from the inflammatory focus and stimulated with Zymosan, they were able to produce HNE in vitro depending on the time of isolation. The highest production of HNE (0.17 μM) by phagocyting neutrophils was observed at the shortest inflammation time studied (3 hrs). In order to compare these results with the oxidative burst of neutrophils the formation of superoxide was also measured by the cytochrome c reduction assay in vitro. The maximum of the production rate of superoxide anion was observed at the same inflammation time (6 hrs), when the HNE maximum occurred. Cells which ingressed earliest (at 3 hrs) showed the highest production rate of superoxide per cell (307 × 10^-18 moles per cell and 30min).

The ability of HNE to attract neutrophils in vivo was studied by adding synthetic HNE to the Sephadex gel and measuring the ingression of neutrophils afterwards. The application of 1 μM HNE in the focus did not change the number of neutrophils but 10 μM HNE increased the cell number by a factor of 3.

The results indicate that HNE is not only a chemoattractant for rat neutrophils in vitro but also in vivo. It is suggested that HNE is produced by selfdestruction of neutrophils during a traumatic inflammation and its production seems to be tightly connected to the oxidative burst of neutrophils. The idea of HNE as part of an autocatalytic cycle is supported whereby neutrophils which immigrate into an inflammatory focus produce HNE which stimulates the ingress of new neutrophils.     

Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity

A novel monocyte-derived neutrophil-activating peptide (MONAP) produced by lipopolysaccharide- and phorbol myristate acetate-stimulated human peripheral blood monocytes was purified by sequential ion exchange-high performance liquid chromatography (HPLC), size exclusion HPLC, and reversed phase HPLC. Biologic activities of the purified cytokine were monitored by either an enzyme release assay or a chemotaxis assay, using peripheral human neutrophils. Purified MONAP was found to be homogeneous, giving a single peak on size-exclusion HPLC, reversed-phase HPLC, as well as a single 10-kDa band on silver-stained polyacrylamide gels. Purified MONAP stimulate human neutrophil chemotaxis at an estimated molarity of 5 x 10(-11) M. Half-maximal enzyme release of cytochalasin B pretreated neutrophils occurred at 2 to 3 x 10(-10) M, whereas superoxide anion production elicited by various concentrations of MONAP was found to be low. Isolated human peripheral monocytes, as well as human eosinophils, showed no chemotactic response to MONAP, indicating neutrophil specificity. MONAP activity was separated from thymocyte-stimulating activity by reversed-phase HPLC, indicating nonidentity with interleukin (IL)-1. This was further supported by heat resistance of MONAP, which is in contrast to the heat sensitivity of IL-1. In addition, IL-1 obtained as a by-product during isolation of MONAP did not stimulate human neutrophil chemotaxis.     

The role of lipid peroxidation and myeloperoxidase in priming a respiratory burst in neutrophils under the action of combined constant and alternating magnetic fields

The enhancement of lipid peroxidation in neutrophils (the content of malonic dialdehyde increased by 10.2%) has been shown after a 1-h exposure to a combined constant (42 μT) magnetic field and a weak low-frequency magnetic field (1.0, 4.4, and 16.5 Hz; 860 nT) collinear to it. No correlation was found between this effect and the process of functional pre-activation (priming) of neutrophils as a result of the combined action of magnetic fields detected by chemiluminescence enhancement in response to the introduction of the bacterial peptide N-formyl–Met–Leu–Phe in the presence of luminol, since ionol (10 μM), an inhibitor of lipid peroxidation, did not reduce the neutrophil priming index in this case. Preliminary addition of histidine (0.1 and 1.0 mM), a singlet oxygen scavenger, also did not decrease the priming index. A myeloperoxidase inhibitor, sodium azide (0.1 mM), exerted a significant inhibitory effect on the chemiluminescence intensity of the neutrophil suspension; priming did not develop in the presence of this inhibitor after the action of combined magnetic fields.     

Icariside II Reduces Testosterone Production by Inducing Necrosis in Rat Leydig Cells

The present study demonstrates that Icariside II (10, 20, and 40 µM) reduced Leydig cell testosterone production and cell viability in a concentration‐ and time‐dependent manner. Hoechst 33342/propidium iodide staining indicated that no morphological changes in Leydig cell nuclear chromatin occurred, caspase‐3 expression also showed no significant change, but cell death was caused by the 10‐µM Icariside II treatment. Furthermore, a significant reduction in NAD⁺ levels was observed following Icariside II exposure (10, 20, and 40 µM). Cell death was avoided when Icariside II treated cells were incubated with extracellular NAD⁺ (5 and 10 mM). Moreover, the addition of NAD⁺ (5 and 10 mM) could restore ATP production and prevent cell death. The results suggest that Icariside II can reduce testosterone production by inducing necrosis, but not apoptosis, in rat Leydig cells. This mechanism may also account for the Icariside II induced depletion of NAD⁺ and ATP levels. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:243‐250, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21481     

Inhibition of osteoblast function in vitro by aminobisphosphonates

Bisphosphonates are analogues of pyrophosphate, a key physicochemical inhibitor of mineralisation. We examined the direct actions of bisphosphonates on the function of cultured osteoblasts derived from rat calvariae. Treatment with zoledronate, the most potent bisphosphonate studied, reduced osteoblast number at concentrations ≥100 nM and was strongly toxic at 10 µM, causing a threefold decrease in osteoblast viability after 2 days and a 90% decrease in cell numbers after 14 days. In control osteoblast cultures on plastic, abundant formation of ‘trabecular’ mineralised bone matrix nodules began after 10 days. Continuous exposure to zoledronate inhibited bone mineralisation at concentrations as low as 10 nM. Pamidronate and clodronate exerted similar effects but at higher doses (≥1 and ≥10 µM, respectively). Short‐term or intermittent exposure of osteoblasts to zoledronate and pamidronate (1–10 µM) was sufficient to inhibit bone mineralisation by ≥85%. Zoledronate but not pamidronate or clodronate also strongly inhibited osteoblast alkaline phosphatase activity at concentrations ≥100 nM and soluble collagen production at concentrations ≥1 µM. We additionally studied the effects of zoledronate on osteoblasts cultured on dentine, a bone‐like mineralised substrate, observing similar inhibitory effects, although at concentrations 10–100‐fold higher; this shift presumably reflected adsorption of zoledronate to dentine mineral. Thus, zoledronate blocked bone formation in two ways: first, a relatively non‐toxic, selective inhibition of mineralisation at concentrations in the low nanomolar range and second, a cytotoxic inhibition of osteoblast growth and function at concentrations ≥1 µM. Although no data are available on the bisphosphonate concentrations that osteoblasts could be exposed to in vivo, our results are consistent with earlier observations that bisphosphonates may inhibit bone formation. J. Cell. Biochem. 106: 109–118, 2009. © 2008 Wiley‐Liss, Inc.