Nitric oxide selectively enhances cAMP levels and electrical coupling between identified RPaD2/VD1 neurons in the CNS of Lymnaea stagnalis (L.)

The isolated CNS of the freshwater mollusc Lymnaea stagnalis was used as a model to study the role of cAMP in NO-mediated mechanisms. The NO donor, DEA/NO (10(-5)-10(-3) M) increased cAMP concentrations in the cerebral, pedal, pleural, parietal and visceral ganglia. In contrast, in the buccal ganglia the same doses of DEA/NO decreased the level of cAMP production. The NOS inhibitor, L-NNA (10(-4) M) increased cAMP concentrations in all areas of the CNS. L-arginine (1 mM), a metabolic precursor of NO, mimicked the action of the NO-donor. The coefficient of electrical coupling between two viscero-parietal peptidergic neurons (VD1/RPaD2) was enhanced by both DEA/NO (10(-4) M) and 8-Br-cAMP (10(-4) M) whereas 8-Br-cGMP (2x10(-4) M) reduced the coupling. We suggest that cAMP-dependent mechanisms are involved in neuronal NO signaling in this simpler nervous system.     

Acute effects of reducing vertical displacement and step frequency on running economy

This work studies the immediate effects of altering the vertical displacement of the center of mass (VD) and step frequency (SF) on the metabolic cost of level treadmill running at 16 km · h(-1) on 16 male runners. Alterations of VD, SF, and the product VD × SF was induced using a novel feedback system, which presents target and current values to the runner by visual or auditory display. Target values were set to 5 and 10% reductions from individual baseline values. The results were expressed as relative changes from baseline values. Alterations led to an increase in metabolic cost in most cases, measured as V(O2) uptake per minute and kilogram of body mass. Correlations were weak. Still, linear multiple regression revealed a positive coefficient (0.28) for the relationship between VD × SF and V(O2). Separate rank correlation tests showed negative correlation (τ = -0.19) between SF and V(O2) and positive correlation (τ = 0.16) between VD and V(O2). There is a coupling between VD and SF caused by the mechanics of running; hence, isolated reduction of either factor was hard to achieve. The linear model also showed a negative coefficient for the relationship between the height of the center of mass above the ground (CoMh) and V(O2). The effect size was small (multiple R(2) 0.07 and 0.12). Still the results indicate that reducing VD × SF by reducing the vertical displacement can have a positive effect on running economy, but a concurrent reduction in CoMh may diminish the positive effect. Midterm and long-term effects of altering the technique should also be studied.     

Effect of acute temperature change on lung respiration of the mollusc Lymnaea stagnalis

• 1.Temperature-dependent effects on respiratory behaviour as well as the corresponding temperature-dependent activities of identified neurons within the respiratory network of the pulmonate snail Lymnaea stagnalis were investigated.
• 2.Lymnaea lung ventilation terminated at low temperatures (under 10 °C) while temperature elevation increased ventilation rates. The respiratory central pattern generator (CPG) functioning was relatively quiescent at temperatures under 12.5±0.44 °C.
• 3.Identified CPG neurons (RPeD1, VD4, VD1/RPaD2) and the respiratory network motor neurons (Vi- and RPa-cells) were found to exhibit varied temperature-dependent electrophysiological parameters (action potential frequency and amplitude, resting potential value) between cell types.
• 4.The observed alterations in the electrical activity of the Lymnaea respiratory network neurons underlie the marked changes of respiratory behaviour observed in the intact animal during temperature changes.

     

Electrical Activity of Identified Neurons in the Central Nervous System of a Mollusk Lymnaea stagnalis under Acute Hyperglycemia

Journal of Evolutionary Biochemistry and Physiology - Rapid responses of the key interneurons identified in the respiratory (RPeD1), locomotor (LPeD1) and cardioregulatory (VD1/RPaD2) networks of...     

The Glutathione System of Peroxide Detoxification Is Less Efficient in Neurons than in Astroglial Cells

The ability of neurons to detoxify exogenously applied peroxides was analyzed using neuron-rich primary cultures derived from embryonic rat brain. Incubation of neurons with H2O2 at an initial concentration of 100 microM (300 nmol/3 ml) led to a decrease in the concentration of the peroxide, which depended strongly on the seeding density of the neurons. When 3 x 10(6) viable cells were seeded per dish, the half-time for the clearance by neurons of H2O2 from the incubation buffer was 15.1 min. Immediately after application of 100 microM H2O2 to neurons, glutathione was quickly oxidized. After incubation for 2.5 min, GSSG accounted for 48% of the total glutathione. Subsequent removal of H2O2 caused an almost complete regeneration of the original ratio of GSH to GSSG within 2.5 min. Compared with confluent astroglial cultures, neuron-rich cultures cleared H2O2 more slowly from the incubation buffer. However, if the differences in protein content were taken into consideration, the ability of the cells to dispose of H2O2 was identical in the two culture types. The clearance rate by neurons for H2O2 was strongly reduced in the presence of the catalase inhibitor 3-aminotriazol, a situation contrasting with that in astroglial cultures. This indicates that for the rapid clearance of H2O2 by neurons, both glutathione peroxidase and catalase are essential and that the glutathione system cannot functionally compensate for the loss of the catalase reaction. In addition, the protein-normalized ability of neuronal cultures to detoxify exogenous cumene hydroperoxide, an alkyl hydroperoxide that is reduced exclusively via the glutathione system, was lower than that of astroglial cells by a factor of 3. These results demonstrate that the glutathione system of peroxide detoxification in neurons is less efficient than that of astroglial cells.     

Biomolecule-stabilized Au nanoclusters as a fluorescence probe for sensitive detection of glucose

In this work, biomolecule-stabilized Au nanoclusters were demonstrated as a novel fluorescence probe for sensitive and selective detection of glucose. The fluorescence of Au nanoclusters was found to be quenched effectively by the enzymatically generated hydrogen peroxide (H(2)O(2)). By virtue of the specific response, the present assay allowed for the selective determination of glucose in the range of 1.0×10(-5) M to 0.5×10(-3) M with a detection limit of 5.0×10(-6) M. The absorption spectroscopy, X-ray photoelectron spectroscopy (XPS) and fluorescence decay studies were then performed to discuss the quenching mechanism. In addition, we demonstrated the application of the present approach in real serum samples, which suggested its great potential for diagnostic purposes.     

Gold-coated silica-fiber hybrid materials for application in a novel hydrogen peroxide biosensor

We describe the preparation and characterization of a novel type of core-shell hybrid material for application in a novel hydrogen peroxide biosensor, where the structure consists of a continuous gold shell that encapsulates the silica fiber. The SiO(2)@Au nanofibers had been synthesized by electrospinning silica sol, and then golden seeds were in situ grown on the fiber, lastly the gold-seeded silica fibers were further coated by continuous gold shells. The above nanocomposites had satisfactory chemical stability, excellent biocompatibility and efficient electron transfer property, which may have potential application for the highly sensitive chemical or biological sensors. Cyclic voltammetry (CV) was used to evaluate the electrochemical performance of the SiO(2)@Au nanocomposites at indium tin oxide (ITO). The biosensor showed high sensitivity and fast response upon the addition of H(2)O(2) and the linear range to H(2)O(2) was from 5×10(-6) to 1.0×10(-3)M with a detection limit of 2 μM (S/N=3). The apparent Michaelis-Menten constant of the biosensor was 1.11 mmol L(-1). These results indicated that SiO(2)@Au nanocomposites have potential for constructing of a variety of electrochemical biosensors.     

Effects of met-enkephalin on electrical coupling between identified neurons in the pulmonate snails,Helix and Lymnaea

1. The effects of met-enkephalin on electrical coupling between molluscan neurons have been investigated using the isolated brains of Helix pomatia and Lymnaea stagnalis.2. In the presence of both serotonin and met-enkephalin, non-rectifying electrical coupling is strongly facilitated between identified respiratory neurons in Helix, whilst coupling between putative, serotonin-containing, ciliomotoneurons in Lymnaea is facilitated by met-enkephalin alone.3. Facilitation of coupling by met-enkephalin is weaker in the strongly coupled neurons, VDl/RPaD2 of Lymnaea.4. These data suggest that met-enkephalin can modulate different groups of electrically coupled cells and may be involved in coordination of motor patterns.     

The Positive Effect of Exogenous Hemin on a Resistance of Strict Anaerobic Archaeon Methanobrevibacter arboriphilus to Oxidative Stresses

Methanogenic archaeon Methanobrevibacter arboriphilus (strains AZ and DH1), which is a strict anaerobic microorganism not able to synthesize heme, possessed a very high catalase activity in the presence of 20-50?μM hemin in a growth medium. We investigated the effect of various oxidative stresses (hydrogen peroxide and oxygenation) on M. arboriphilus cells grown on the standard nutrient medium supplemented with 0.1?% yeast extract, and on the same medium supplemented with hemin. It was demonstrated that 30?μM hemin had a very significant positive effect on the resistance of M. arboriphilus strains to H(2)O(2) and O(2) stresses because of 30- to 40-fold increase of heme catalase activity. Thus, hydrogen peroxide (0.6-1.2?mM) or oxygen (3-5?%) had a strong negative impact on low-catalase cultures grown in the hemin-free standard medium, whereas the presence of 30?μM hemin in the medium results in a high yield of biomass even under conditions of four times stronger H(2)O(2) and two times stronger O(2) stresses. The intracellular catalase activity reached a high level in 30-60?min after hemin was added to the nutrient medium, but the activity already increased about 5-7-fold in 6?min after hemin addition. Our experimental data suggest that exogenous hemin provides an effective antioxidative defense in representatives of the genus Methanobrevibacter, specially playing an important role in the puromycin-insensitive formation of the active heme-containing catalase from presynthesized apoenzyme and heme.     

The effect of hydrogen peroxide on the growth of microscopic mycelial fungi isolated from habitats with different levels of radioactive contamination

The effect of hydrogen peroxide ( 10(-9)-10(-1) M) on the mycelial growth of the fungi Alternaria alternata, Cladosporium cladosporioides, Mucor hiemalis, and Paecilomyces lilacinus has been studied. The growth of fungi isolated from habitats with a background level of radioactive contamination was stopped by H2O2 concentrations equal to 10(-3) and 10(-2) M, whereas the growth of fungi that were isolated from habitats with high levels of radioactive contamination was only arrested by 10(-1) M H2O2. The response of the different fungi to hydrogen peroxide was of three types: (1) a constant growth rate of fungal hyphae at H2O2 concentrations between 10(-9) and 10(-4) M and a decrease in this rate at 10(-3) M H2O2, (2) a gradual decrease in the growth rate as the H2O2 concentration was increased, and (3) an increase in the growth rate as the H2O2 concentration was increased from 10(-7) to 10(2)-5 M. The melanin-containing species A. alternata and C. cladosporioides exhibited all three types of growth response to hydrogen peroxide, whereas the light-pigmented species M. hiemalis and P. lilacinus showed only the first type of growth response. A concentration of hydrogen peroxide equal to 10(-1) M was found to be lethal to all of the fungi studied. The most resistant to hydrogen peroxide was found to be the strain A. alternata 56, isolated from the exclusion zone of the Chernobyl Nuclear Power Plant.     

Electrospun polystyrene-poly(styrene-co-maleic anhydride) nanofiber as a new aptasensor platform

Here we report on a new approach for the electrochemical detection of hydrogen peroxide (H(2)O(2)) based on the co-immobilization of horseradish peroxidase and methylene blue on the functionalized carbon buckypaper supported by a titanium substrate. Cyclic voltammetry was used to study and optimize the performance of the resulting electrochemical biosensor. The proposed biosensor exhibited high analytical performance towards the quantification of H(2)O(2) at the physiological pH 7.4. Under optimized conditions, the biosensor shows a wide linear response range from 0.1 × 10(-6) to 5 × 10(-4)M concentrations of H(2)O(2). The detection limit was determined to be 7.5 × 10(-8)M (based on S/N=3). Reproducibility and stability of the fabricated biosensor were examined with satisfactory results. The biological relevance of the developed electrochemical biosensor has been further studied by the determination of H(2)O(2) in human urine samples of normal volunteers prior to and following the ingestion of coffee. Increased levels of urinary H(2)O(2) concentration suggest that oxidative stress is induced by coffee drinking in humans. There is considerable interest in oxidative stress as relates to human physiology. The sensitive determination of H(2)O(2) in human urine may serve as a valuable biomarker to effectively elucidate specific levels of oxidative stress in vivo.     

Sensitivity to low-dose/low-LET ionizing radiation in mammalian cells harboring mutations in succinate dehydrogenase subunit C is governed by mitochondria-derived reactive oxygen species

It has been hypothesized that ionizing radiation-induced disruptions in mitochondrial O? metabolism lead to persistent heritable increases in steady-state levels of intracellular superoxide (O?(?U+2212)) and hydrogen peroxide (H?O?) that contribute to the biological effects of radiation. Hamster fibroblasts (B9 cells) expressing a mutation in the gene coding for the mitochondrial electron transport chain protein succinate dehydrogenase subunit C (SDHC) demonstrate increases in steady-state levels of O??- and H?O?. When B9 cells were exposed to low-dose/low-LET radiation (5-50 cGy), they displayed significantly increased clonogenic cell killing compared with parental cells. Clones derived from B9 cells overexpressing a wild-type human SDHC (T4, T8) demonstrated significantly increased surviving fractions after exposure to 5-50 cGy relative to B9 vector controls. In addition, pretreatment with polyethylene glycol-conjugated CuZn superoxide dismutase and catalase as well as adenoviral-mediated overexpression of MnSOD and/or mitochondria-targeted catalase resulted in significantly increased survival of B9 cells exposed to 10 cGy ionizing radiation relative to vector controls. Adenoviral-mediated overexpression of either MnSOD or mitochondria-targeted catalase alone was equally as effective as when both were combined. These results show that mammalian cells over expressing mutations in SDHC demonstrate low-dose/low-LET radiation sensitization that is mediated by increased levels of O??- and H?O?. These results also support the hypothesis that mitochondrial O??- and H?O? originating from SDH are capable of playing a role in low-dose ionizing radiation-induced biological responses.     

A novel enzymatic hydrogen peroxide biosensor based on Ag/C nanocables

A novel enzymatic hydrogen peroxide sensor was successfully fabricated based on the nanocomposites containing of Ag/C nanocables and gold nanoparticles (AuNPs). Ag/C nanocables have been synthesized by a hydrothermal method and then AuNPs were assembled on the surface of Ag/C nanocables. The nanocomposites were confirmed by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometry (EDS). The above nanocomposites have satisfactory chemical stability and excellent biocompatibility. Cyclic voltammetry (CV) was used to evaluate the electrochemical performance of the Ag/C/Au nanocomposites at glassy carbon electrode (GCE). The results indicated that the Ag/C/Au nanocomposites exhibited excellent electrocatalytic activity to the reduction of H(2)O(2). It offered a linear range of 6.7×10(-9) to 8.0×10(-6) M, with a detection limit of 2.2×10(-9) M. The apparent Michaelis-Menten constant of the biosensor was 51.7×10(-6) M. These results indicated that Ag/C/Au nanocomposites have potential for constructing of a variety of electrochemical biosensors.     

Oxytocin hyperpolarizes cultured duodenum myenteric intrinsic primary afferent neurons by opening BK(Ca) channels through IP₃ pathway

Oxytocin (OT) is clinically important in gut motility and constitutively reduces duodenum contractility. Intrinsic primary afferent neurons (IPANs), whose physiological classification is as AH cells, are the 1st neurons of the peristaltic reflex pathway. We set out to investigate if this inhibitory effect is mediated by IPANs and to identify the ion channel(s) and intracellular signal transduction pathway that are involved in this effect. Myenteric neurons were isolated from the longitudinal muscle myenteric plexus (LMMP) preparation of rat duodenum and cultured for 16-24 h before electrophysiological recording in whole cell mode and AH cells identified by their electrophysiological characteristics. The cytoplasmic Ca2? concentration ([Ca2?](i) ) of isolated neurons was measured using calcium imaging. The concentration of IP(3) in the LMMP and the OT secreted from the LMMP were measured using ELISA. The oxytocin receptor (OTR) and large-conductance calcium-activated potassium (BK(Ca)) channels, as well as the expression of OT and the IPAN marker calbindin 28 K, on the myenteric plexus neurons were localized using double-immunostaining techniques. We found that administration of OT (10?? to 10?? M) dose dependently hyperpolarized the resting membrane potential and increased the total outward current. The OTR antagonist atosiban or the BK(Ca) channel blocker iberiotoxin (IbTX) blocked the effects of OT suggesting that the increased outward current resulted from BK(Ca) channel opening. OTR and the BK(Ca) α subunit were co-expressed on a subset of myenteric neurons at the LMMP. NS1619 (10?? M, a BK(Ca) channel activator) increased the outward current similar to the effect of OT. OT administration also increased [Ca2?](i) and the OT-evoked outward current was significantly attenuated by thapsigargin (10?? M) or CdCl?. The effect of OT on the BK(Ca) current was also blocked by pre-treatment with the IP? receptor antagonist 2-APB (10?? M) or the PLC inhibitor U73122 (10?? M). OT (10?? M) also increased the IP? concentration within the LMMP. Both of the spontaneous and KCl-induced secretion of OT was enhanced by atosiban. Most of OT-immunoreactive cells are also immunoreactive for calbindin 28 K. In summary, we concluded that OT hyperpolarized myenteric IPANs by activating BK(Ca) channels via the OTR-PLC-IP?-Ca2? signal pathway. OT might modulate IPANs mediated ENS reflex by an autocrine and negative feedback manner.     

Regulation of T-cell activation and T-cell growth factor (TCGF) production by hydrogen peroxide

Activated macrophages are known to release a variety of immunoregulatory substances including the low-molecular-weight substances hydrogen peroxide and lactate. We report here that lactate but not hydrogen peroxide is capable of supporting a substantial production of T-cell growth factor (TCGF) in cultures of accessory cell-depleted splenic T-cell populations after stimulation with concanavalin A. Hydrogen peroxide and its biosynthetic precursor superoxide anion (O2-) mediate, however, a strong augmentation of the TCGF production by accessory cell-depleted T-cell populations in the presence of lactate. Lactate inhibits the incorporation of [3H]thymidine in short-term cultures (18-26 hr) of accessory cell-depleted T cells. This confirms the rule that (optimal) production of T-cell growth factor requires a growth inhibitory signal. Concentrations of hydrogen peroxide which augment TCGF production most effectively (i.e., 1 X 10(-5) M) do not inhibit the incorporation of [3H]thymidine; and higher concentrations (3 X 10(-5)-1 X 10(-4) M) of hydrogen peroxide inhibit both the production of TCGF and the incorporation of [3H]thymidine. In agreement with the augmenting effect of hydrogen peroxide on TCGF production, it was observed that the proliferative response in mixed lymphocyte cultures is suppressed by catalase and augmented by 1 X 10(-5) M H2O2. Proliferative and cytotoxic responses in mixed lymphocyte cultures with an external source of interleukin 2 (IL-2) in contrast, are not augmented by 1 X 10(-5) M H2O2. The relatively high concentration of 1 X 10(-4) M hydrogen peroxide was found to inhibit the proliferative responses in mixed lymphocyte cultures with or without external IL-2 but not the cytotoxic response in the presence of IL-2. This indicates that CTL precursor cells may be relatively resistant against H2O2.     

The Haber-Weiss cycle--70 years later

The chain reactions HO* + H2O2 --> H2O + O2*- + H+ and O2*- + H+ + H2O2 --> O2 + HO* + H2O, commonly known as the Haber-Weiss cycle, were first mentioned by Haber and Willst?tter in 1931. George showed in 1947 that the second reaction is insignificant in comparison to the fast dismutation of superoxide, and this finding appears to have been accepted by Weiss in 1949. In 1970, the Haber-Weiss reaction was revived by Beauchamp and Fridovich to explain the toxicity of superoxide. During the 1970s various groups determined that the rate constant for this reaction is of the order of 1 M(-1) s(-1) or less, which confirmed George's conclusion. The reaction of superoxide with hydrogen peroxide was dropped from the scheme of oxygen toxicity, and superoxide became the source of hydrogen peroxide, which yields hydroxyl radicals via the Fenton reaction, Fe2+ + H2O2 --> Fe3+ + HO- + HO*. In 1994, Kahn and Kasha resurrected the Haber-Weiss reaction again, but this time the oxygen was believed to be in the singlet (1delta(g)) state. As toxicity arises not from a Fenton-catalysed Haber-Weiss reaction, but from the Fenton reaction, the Haber-Weiss reaction should not be mentioned anymore.     

The effect of iron-chelating agents on Magnetospirillum magneticum strain AMB-1: stimulated growth and magnetosome production and improved magnetosome heating properties

The introduction of various iron-chelating agents to the Magnetospirillum magneticum strain AMB-1 bacterial growth medium stimulated the growth of M. magneticum strain AMB-1 magnetotactic bacteria and enhanced the production of magnetosomes. After 7?days of growth, the number of bacteria and the production of magnetosomes were increased in the presence of iron-chelating agents by factors of up to ??2 and ??6, respectively. The presence of iron-chelating agents also produced an increase in magnetosome size and chain length and yielded improved magnetosome heating properties. The specific absorption rate of suspensions of magnetosome chains isolated from M. magneticum strain AMB-1 magnetotactic bacteria, measured under the application of an alternating magnetic field of average field strength ??20?mT and frequency 198?kHz, increased from ??222?W/g_Fe in the absence of iron-chelating agent up to ??444?W/g_Fe in the presence of 4???M rhodamine B and to ??723?W/g_Fe in the presence of 4???M EDTA. These observations were made at an iron concentration of 20???M and iron-chelating agent concentrations below 40???M.     

Evidence for nonhydrogen bonded compound II in cyclic reaction of hemoglobin I from Lucina pectinata with hydrogen peroxide

Studies that elucidate the behavior of the hemoglobins (Hbs) and myoglobins upon reaction with hydrogen peroxide are essential to the development of oxygen carrier substitutes. Stopped-flow kinetics and resonance Raman data show that the reaction between hydrogen peroxide and oxygenated and deoxygenated ferric Hb I (oxy- and deoxy-HbI) from Lucina pectinata produce compound I and compound II ferryl species. The rate constants ratio (k23/k41) between the formation of compound II from compound I (k23) and the oxidation of the ferrous HbI (k41, i.e., 25 M(-1) s(-1)) of 12 x 10(-4) M suggests that HbI has a peroxidative capacity for removing H2O2 from solution. Resonance Raman presents the formation of both, met-aquo-HbI and compound II ferryl species in the cyclic reaction of HbI with H2O2. The ferric HbI species is maintained by the presence of H2O2; it can produce HbI compound I, or it can be reduced to a deoxy-HbI derivative to form HbI compound II upon reaction with H2O2. The compound II ferryl vibration frequency appears at 805 and 769 cm(-1) for HbIFe(IV)=(16)O and HbIFe(IV)=(18)O species, respectively. This ferryl mode indicates the absence of hydrogen bonding between the carbonyl group of the distal Q64 and the HbIFe(IV)=O ferryl moiety. The observation suggests that both the trans-ligand effect and the polarizabilty of the HbI heme pocket are responsible for the observed ferryl oxo vibrational energy. The vibrational mode also suggests that the carbonyl group of the distal Q64 is oriented toward the iron of the heme group, increasing the distal pocket electron density.     

Preventive and protective effects of Turkish propolis on H₂O₂-induced DNA damage in foreskin fibroblast cell lines

The aim of the present study was to evaluate the potential of Turkish propolis extracts if they prevent or protect foreskin fibroblast cells against hydrogen peroxide (H?O?)-induced oxidative DNA damage. Hydrogen peroxide (40 μM) was used as an inducer of oxidative DNA damage. The damage of DNA was evaluated by using the alkaline single cell gel electrophoresis (comet) assay. Turkish propolis extracts at concentrations of 25, 50, 75 and 100 μg/ml were prepared by ethanol. Anti-genotoxicity was assessed before, simultaneously, and after treatment of propolis extract (50 μg/ml) with H?O?. The results showed a significant decrease in H?O?-induced DNA damage in cultures treated with propolis extract. The antioxidant activity of phenolic components found in propolis may contribute to reduce the DNA damage induced by H?O?. Our findings confirmed the chemopreventive activity of propolis and showed that this effect may occur under different mechanisms.