Is hydrogen peroxide produced during iron(II) oxidation in mammalian apoferritins?

The ferritins are a class of iron storage and detoxification proteins that play a central role in the biological management of iron. These proteins have a catalytic site, "the ferroxidase site", located on the H-type subunit that facilitates the oxidation of Fe(II) to Fe(III) by O(2). Measurements during the past 10 years on a number of vertebrate ferritins have provided evidence that H(2)O(2) is produced at this diiron ferroxidase site. Recently reported experiments using three different analytical methods with horse spleen ferritin (HoSF) have failed to detect H(2)O(2) production in this protein [Lindsay, S., Brosnahan, D., and Watt, G. D. (2001) Biochemistry 40, 3340-3347]. These findings contrast with earlier results reporting H(2)O(2) production in HoSF [Xu, B., and Chasteen, N. D. (1991) J. Biol. Chem. 266, 19965-19970]. Here a sensitive fluorescence assay and an assay based on O(2) evolution in the presence of catalase were used to demonstrate that H(2)O(2) is produced in HoSF as previously reported. However, because of the relatively few H-chain ferroxidase sites in HoSF and the reaction of H(2)O(2) with the protein, H(2)O(2) is more difficult to detect in this ferritin than in recombinant human H-chain ferritin (HuHF). The proper sequence of addition of reagents is important for measurement of the total amount of H(2)O(2) produced during the ferroxidation reaction.     

Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance?

Elevated levels of salicylic acid (SA) are required for the induction of systemic acquired resistance (SAR) in plants. Recently, a salicylic acid-binding protein (SABP) isolated from tobacco was shown to have catalase activity. Based on this finding elevated levels of hydrogen peroxide (H₂O₂) were postulated to act as a second messenger of SA in the SAR signal transduction pathway. A series of experiments have been carried out to clarify the role of H₂O₂ in SAR-signaling. No increase of H₂O₂ was found during the onset of SAR. Induction of the SAR gene, PR-1, by H₂O₂ and H₂O₂-inducing chemicals is strongly suppressed in transgenic tobacco plants that express the bacterial salicylate hydroxylase gene, indicating that H₂O₂ induction of SAR genes is dependent on SA accumulation. Following treatment of plants with increasing concentrations of H₂O₂, a dose-dependent accumulation of total SA species was found, suggesting that H₂O₂ may induce PR-1 gene expression through SA accumulation. While the results do not support a role for H₂O₂ in SAR signaling, it is suggested that SA inhibition of catalase activity may be important in tissues undergoing a hypersensitive response.     

Is there an interleukin 2 inhibitor in human serum?

Normal mouse serum has been shown to contain an inhibitor of interleukin 2 (IL-2). Here we report that a molecule with similar activity cannot be found in normal human serum (NHS). Although NHS inhibited the IL-2-dependent proliferation of mouse CTLL cells, as expected of an IL-2 inhibitor, it also had inhibitory activity on IL-3-dependent cells and was cytolytic to IL-2-independent mouse cells as measured by a ⁵¹Cr release assay, indicating a nonspecific effect. In addition, NHS had no effect on the IL-2-dependent proliferation of human peripheral blood T-cell blasts. Fractionation of NHS by size exclusion HPLC failed to separate cytolytic activity from any putative true IL-2 inhibitor activity. The cytolytic component was not related to immunoglobulin since it had a molecular weight of 50,000 to 60,000 and was not bound by protein-A-Sepharose. However, its molecular weight, heat lability, and trypsin sensitivity suggest it to be a protein.     

Dexamethasone-mediated regulation of death and differentiation of muscle cells. Is hydrogen peroxide involved in the process?

The hypothetical involvement of H2O2 in dexamethasone-mediated regulation of muscle cell differentiation and elimination was studied. Rat L6 myoblasts and mouse C2C12 satellite cells were chosen for acute (24 h) and chronic (5 or 10 day) experiments. Mitogenicity and anabolism were both affected by H2O2. Micromolar concentrations of H2O2 inhibited DNA while stimulating protein synthesis. At the millimolar level, H2O2 led to cell death by apoptosis.Synthetic glucocorticoi - dexamethasone (Dex) was shown to effect muscle cell fate similarly to H2O2. Chronic treatment with H2O2 or Dex dose-dependently accelerated either the formation of myotubes or cell elimination. Dex-induced cell death slightly differed from classical apoptosis and was featured by the symptoms of cell senescence such as extensive cytoplasm vacuolisation, accumulation of inclusion-bodies and lack of low molecular weight oligonucleosomal DNA fragmentation but chromatin condensation. Antioxidants (sodium ascorbate, N-acetyl-L-cysteine, catalase) abrogated Dex-dependent cell death. We conclude that H2O2 directly influences myogenesis and muscle cell elimination. Moreover, H2O2 can be considered as the potent mediator of glucocorticoid-dependent effects on muscle cells.     

Is the human trophoblast in vitro an adaptive tissue for immunological influences?

The role of the extra-embryonic organs (e.g. placenta) in the feto-maternal immunological interactions is not clear. Trophoblast cells of human placenta were studied in vitro in relation to their adaptive capacities under the immunological influence of allogeneic leucocytes of pregnant women. It was shown that after an 18-h interaction some trophoblastic cells were still viable, they were rich in glycoproteins and RNA, and elicited a prominent activity of some enzymes. Mitosis was often seen. The authors suppose that the trophoblast is highly adaptable to some immunological influences in vitro.     

Formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Is haemoglobin a biological Fenton reagent?

The ability of oxyhaemoglobin and methaemoglobin to generate hydroxyl radicals (OH.) from H2O2 has been investigated using deoxyribose and phenylalanine as 'detector molecules' for OH.. An excess of H2O2 degrades methaemoglobin, releasing iron ions that react with H2O2 to form a species that appears to be OH.. Oxyhaemoglobin reacts with low concentrations of H2O2 to form a 'reactive species' that degrades deoxyribose but does not hydroxylate phenylalanine. This 'reactive species' is less amenable to scavenging by certain scavengers (salicylate, phenylalanine, arginine) than is OH., but it appears more reactive than OH. is to others (Hepes, urea). The ability of haemoglobin to generate not only this 'reactive species', but also OH. in the presence of H2O2 may account for the damaging effects of free haemoglobin in the brain, the eye, and at sites of inflammation.     

Do nitroxides protect cardiomyocytes from hydrogen peroxide or superoxide?

The aim of the research was to study the role played by extracellular O ₂ ^.- radicals, which are implicated in cardiac cell damage and the protective effect by cell-permeable, nitroxide, superoxide dismutase-mimics. Cardiomyocytes cultures from 1-day-old rats served as the test-system. Experiments were performed since 5th day in culture when >80% of the cells were beating myocardial cells. Oxidative damage was induced by 0.5 mM hypoxanthine and 0.06 U/ml xanthine oxidase or by 10 mM glucose and 0.15 U/ml glucose oxidase. The parameters used to evaluate damages were spontaneous beating, lactate dehydrogenase release and ATP level. The rhythmic pulsation was followed microscopically. To determine the kinetics of cytosolic enzyme release from the cells, media samples were collected at various points of time and assayed for enzyme activity. To determine the cellular ATP, cells were washed with sodium phosphate buffer, scraped off and boiled for 3 min with sodium phosphate buffer. Following centrifugation the supernatant was collected and ATP was determined by the chemiluminogenic assay using firefly tails. The present results indicate that nitroxide stable free radicals, in the millimolar concentration range, provide full protection without toxic side-effect. Unlike exogenously added SOD that failed to protect, exogenous catalase provided almost full protection. In addition, the metal-chelating agent dipyridyl, but not diethylene-triamine-pentaacetate or desferrioxamine, protected the cultured cells. The present results suggest that H₂O₂ is the predominant toxic species mediating the oxidative damage whereas extracellular superoxide radical does not contribute to cultured cardiomyocyte damage. Since nitroxides do not remove H₂O₂ they can protect the cells possibly by oxidizing the metal ions and inhibiting the Fenton reaction. The superoxide dismutase-mimic activity of nitroxides does not seem to underlie their protective effect, however, the involvement of intracellular O ₂ ^.- cannot be excluded.Abbreviations CHDO 2-spirocyclohexane doxyl (2-cyclohexane-5,5-demethyl-3-oxazolidinoxyl) - DF desferrioxamine - DTPA diethylene-triamine-pentaacetate - EPR electron paramagnetic resonance - HX hypoxanthine - LDH lactate dehydrogenase - SOD superoxide dismutase - SEM standard error of mean: TEMPOL, 4-hydroxy-2,2,6,6-tetramethyl-piperidinoxyl - TEMPAMINE 4-amino-2,2,6,6-tetramethyl-piperidinoxyl - XO xanthine oxidase - CAT catalase     

Stimulation of β-carotene synthesis by hydrogen peroxide in Blakeslea trispora

-Carotene synthesis was increased from a negligible amount to 152 mg (g-dry cells)^–1 and H₂O₂ was accumulated up to 16.7 M during 2.5 day-culture of Blakeslea trispora. When cells were cultivated in 250 ml flasks containing various volumes (25–150 ml) of the medium, not only H₂O₂ accumulation but also -carotene synthesis increased as culture volume decreased. Addition of H₂O₂ (10 M) to the 1.5-day old cultures of B. trispora resulted in 46% higher -carotene synthesis than that without addition. All these results indicate that -carotene biosynthesis is stimulated by H₂O₂ in B. trispora.     

Is avoiding an aversive outcome rewarding? Neural substrates of avoidance learning in the human brain

Avoidance learning poses a challenge for reinforcement-based theories of instrumental conditioning, because once an aversive outcome is successfully avoided an individual may no longer experience extrinsic reinforcement for their behavior. One possible account for this is to propose that avoiding an aversive outcome is in itself a reward, and thus avoidance behavior is positively reinforced on each trial when the aversive outcome is successfully avoided. In the present study we aimed to test this possibility by determining whether avoidance of an aversive outcome recruits the same neural circuitry as that elicited by a reward itself. We scanned 16 human participants with functional MRI while they performed an instrumental choice task, in which on each trial they chose from one of two actions in order to either win money or else avoid losing money. Neural activity in a region previously implicated in encoding stimulus reward value, the medial orbitofrontal cortex, was found to increase, not only following receipt of reward, but also following successful avoidance of an aversive outcome. This neural signal may itself act as an intrinsic reward, thereby serving to reinforce actions during instrumental avoidance.     

Are respiratory enzymes the primary sources of intracellular hydrogen peroxide?

Endogenous H2O2 is believed to be a source of chronic damage in aerobic organisms. To quantify H2O2 formation, we have generated strains of Escherichia coli that lack intracellular scavenging enzymes. The H2O2 that is formed within these mutants diffuses out into the medium, where it can be measured. We sought to test the prevailing hypothesis that this H2O2 is primarily generated by the autoxidation of redox enzymes within the respiratory chain. The rate of H2O2 production increased when oxygen levels were raised, confirming that H2O2 is formed by an adventitious chemical process. However, mutants that lacked NADH dehydrogenase II and fumarate reductase, the most oxidizable components of the respiratory chain in vitro, continued to form H2O2 at normal rates. NADH dehydrogenase II did generate substantial H2O2 when it was when overproduced or quinones were absent, forcing electrons to accumulate on the enzyme. Mutants that lacked both NADH dehydrogenases respired very slowly, as expected; however, these mutants showed no diminution of H2O2 excretion, suggesting that H2O2 is primarily formed by a source outside the respiratory chain. That source has not yet been identified. In respiring cells the rate of H2O2 production was approximately 0.5% the rate of total oxygen consumption, with only modest changes when cells used different carbon sources.     

Does hydrogen peroxide exist “free” in biological systems?

Hydrogen peroxide (H₂O₂) can diffuse far from the site of production to intracellular locations where biological effects may be greater. The diffusion range is extended by H₂O₂ carriers formed spontaneously by hydrogen bonding with monomeric and polymeric compounds, including amino and dicarboxylic acids, peptides, proteins, nucleic acid bases, and nucleosides. Hydrogen peroxide adducts (HPAs) are readily synthesized, e.g., crystalline histidine (His)-H₂O₂ adducts. An equilibrium exists between an adduct-forming compound and H₂O₂. The detection and relative stabilities of HPAs are measured by the degree of decomposition of H₂O₂ as influenced by test compounds in buffered solution competing with glucose or fructose for H₂O₂. The HPAs delay decomposition of H₂O₂ up to several hundredfold. The overall charge on an HPA, i.e., its ability to penetrate cell membranes, influences the cytotoxic and clastogenic effects of H₂O₂. Growth inhibition of Salmonella typhimurium LT₂ by H₂O₂ is enhanced by neutral HPAs but decreased by anionic HPAs. Addition of catalase 1, 10, or 30 min after inoculation of S. typhimurium LT₂ reduces or nearly eliminates partial growth inhibition by H₂O₂, but a neutral HPA, expecially his-H₂O₂, transported H₂O₂ into the cells within 1 min, and in about 10 min completely inhibited growth. The stability of HPAs decreases with increasing pH or increasing temperature, while added Fe(II) in the presence and absence of EDTA accelerates H₂O₂ and HPA decomposition. Calculations indicate H₂O₂ hydrogen bonds with nucleic acid-base pairs with no apparent bond strain and energy stabilization comparable to normal hydrogen bonding.     

Is sodium current present in human sinoatrial node cells?

Pacemaker activity of the sinoatrial node has been studied extensively in various animal species, but is virtually unexplored in man. As such, it is unknown whether the fast sodium current (I_Na) plays a role in the pacemaker activity of the human sinoatrial node. Recently, we had the unique opportunity to perform patch-clamp experiments on single pacemaker cells isolated from a human sinoatrial node. In 2 out of the 3 cells measured, we observed large inward currents with characteristics of I_Na. Although we were unable to analyze the current in detail, our findings provide strong evidence that I_Na is present in human sinoatrial node pacemaker cells, and that this I_Na is functionally available at potentials negative to -60 mV.     

Protection by α-tocopherol but not ascorbic acid from hydrogen peroxide induced cell death in normal human breast epithelial cells in culture

α-Tocopherol and ascorbic acid have been suggested to play a role in breast cancer prevention due to their antioxidative capacity. Increased exposure to endogenous and exogenous sex steroids is a known risk factor for breast cancer. We have studied the effects of α-tocopherol and ascorbic acid on hydrogen peroxide induced cell death in sex hormone treated normal breast epithelial cells in culture. We found that α-tocopherol but not ascorbic acid alone protected the cells. The effect of α-tocopherol increased when ascorbic acid was added to the cultures. The hydrogen peroxide degradation rate decreased in cultures treated with α-tocopherol alone and in combination with ascorbic acid compared to cells grown in medium or with ascorbic acid only. Oestradiol and progesterone treatment did not influence the results. Possible beneficial effects of combining various antioxidants, endogenous as well as exogenous, on human breast tissue need to be investigated further both in vivo and in vitro.     

Reactivity and endogenous modification by nitrite and hydrogen peroxide: does human neuroglobin act only as a scavenger?

NGB (human neuroglobin), a recently discovered haem protein of the globin family containing a six-co-ordinated haem, is expressed in nervous tissue, but the physiological function of NGB is currently unknown. As well as playing a role in neuronal O2 homoeostasis, NGB is thought to act as a scavenger of reactive species. In the present study, we report on the reactivity of metNGB (ferric-NGB), which accumulates in vivo as a result of the reaction of oxyNGB (oxygenated NGB) with NO, towards NO2- and H2O2. NO2- co-ordination of the haem group accounts for the activity of metNGB in the nitration of phenolic substrates. The two different metNGB forms, with and without the internal disulfide bond between Cys46 (seventh residue on the inter-helix region between helices C and D) and Cys55 (fifth residue on helix D), exhibit different reactivity, the former being more efficient in activating NO2-. The kinetics of the reactions, the NO2--binding studies and the analysis of the nitrated products from different substrates all support the hypothesis that metNGB is able to generate an active species with the chemical properties of peroxynitrite, at pathophysiological concentrations of NO2- and H2O2. Without external substrates, the targets of the reactive species generated by the metNGB/NO2-/H2O2 system are endogenous tyrosine (resulting in the production of 3-nitrotyrosine) and cysteine (oxidized to sulfinic acid and sulfonic acid) residues. These endogenous modifications were characterized by HPLC-MS/MS (tandem MS) analysis of metNGB after reaction with NO2- and H2O2 under various conditions. The internal S-S bond affects the functional properties of the protein. Therefore metNGB acts not only as scavenger of toxic species, but also as a target of the self-generated reactive species. Self-modification of the protein may be related to or inhibit its postulated neuroprotective activity.     

We-intentionality: an essential element in understanding human culture?

Culture is a powerful cause of human behavior, yet there have been few attempts to synthesize the biological and social sciences in order to provide an explanation of culture and hence to lay the foundations for a comprehensive human science. This essay explores one possible way of doing this by outlining the likely psychological mechanisms that give rise to the unique human capacity for creating social reality. The core assumption is that "we-intentionality," which is key to understanding social constructions, can and must be linked to specific evolved psychological traits, and hence placed within a neuroscience perspective.     

Is Chytridiomycosis an Emerging Infectious Disease in Asia?

The disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has caused dramatic amphibian population declines and extinctions in Australia, Central and North America, and Europe. Bd is associated with >200 species extinctions of amphibians, but not all species that become infected are susceptible to the disease. Specifically, Bd has rapidly emerged in some areas of the world, such as in Australia, USA, and throughout Central and South America, causing population and species collapse. The mechanism behind the rapid global emergence of the disease is poorly understood, in part due to an incomplete picture of the global distribution of Bd. At present, there is a considerable amount of geographic bias in survey effort for Bd, with Asia being the most neglected continent. To date, Bd surveys have been published for few Asian countries, and infected amphibians have been reported only from Indonesia, South Korea, China and Japan. Thus far, there have been no substantiated reports of enigmatic or suspected disease-caused population declines of the kind that has been attributed to Bd in other areas. In order to gain a more detailed picture of the distribution of Bd in Asia, we undertook a widespread, opportunistic survey of over 3,000 amphibians for Bd throughout Asia and adjoining Papua New Guinea. Survey sites spanned 15 countries, approximately 36° latitude, 111° longitude, and over 2000 m in elevation. Bd prevalence was very low throughout our survey area (2.35% overall) and infected animals were not clumped as would be expected in epizootic events. This suggests that Bd is either newly emerging in Asia, endemic at low prevalence, or that some other ecological factor is preventing Bd from fully invading Asian amphibians. The current observed pattern in Asia differs from that in many other parts of the world.