Irina A. Slepneva

Influence of fungal infection on the DOPA-semiquinone and DOPA-quinone production in haemolymph of Galleriamellonella larvae.

The formation of reactive oxygen metabolites in haemolymph of Galleria mellonella larvae was studied by ESR spectroscopy. The inhibition of the production of the reactive oxygen metabolites of DOPA in haemolymph under the action of fungal infection was shown using spin trap 1-hydroxy-3-carboxy-pyrrolidine. This inhibition correlated with decrease of phenoloxidase activity in haemolymph of infected insects. Simultaneously, the decrease of production of DOPA-semiquinone was detected using method of spin stabilization by diamagnetic metal ions. Moreover, it was shown that the formation of DOPA-quinone was slowed down in haemolymph of infected insects. Our results suggest that the DOPA-derived quinones/semiquinones may be involved in immune response of insects as part of its defense mechanism.

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase.

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-dl-alanine (DOPA) has been studied. The ability of DOPA to react with has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and being equal to (3.4±0.6)×105 M−1 s−1. The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with

The phenylthiourea is a competitive inhibitor of the enzymatic oxidation of DOPA by phenoloxidase

Phenoloxidase is a key enzyme of melanization catalyzing the oxidation of phenols. Phenylthiourea (PTU) is the well-known and widely used inhibitor of phenoloxidase. However, the mechanism of its action is not quite clear. In the present work, the effect of PTU on the enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-l-alanine (DOPA) by phenoloxidase was studied by spectrophotometric methods. The inhibition constant of PTU was estimated as 0.21 ± 0.09 µM and the competitive type of inhibition was determined for this reaction.

Quantitative determination of nitric oxide production in haemocytes: Nitrite reduction activity as a potential pathway of NO formation in haemolymph of Galleria mellonella larvae

The generation of nitric oxide by Galleria mellonella larvae haemocytes has been investigated. For this purpose, a fluorescent method, specific for detection of NO, has been developed. The method is based on the application of fluorescence probe DAF-FM diacetate and nitronyl nitroxyl radical, NNR, which accelerates the NO-dependent formation of fluorescence product, DAF-FM triazole. The key feature of the method is the registration and analysis of differential kinetics, namely, the difference of kinetics obtained in samples with NNR and without NNR. This approach allows us to exclude any other kinetic processes not related to triazole formation. The differential kinetics were calibrated versus NO generation rate and the resulting low limit of method sensitivity was obtained as about 0.4-0.5 nM/min. The generation of nitric oxide by the haemocytes of insects treated with LPS in vivo has been detected at a rate of 0.5-0.7 nM/min. However, the production of NO in haemocyte suspensions treated with both the substrate, l-arginine, and the inhibitor, l-NAME, of NOS, has not been detected within method sensitivity. These data provide only the upper level of NO generation by haemocytes but cannot be used to draw definite conclusions about NOS as a source of NO. Meanwhile, it is known, that NO can be formed by NOS independent mechanism. Indeed, we have observed a significant increase in NO generation in the samples of haemocytes intracellularly loaded with nitrite. Moreover, adding nitrite to lysed haemocytes results in the higher NO generation rate. After addition of 500 μM nitrite, the rates of NO generation in the samples are determined to be 2 and 20-30 nM/min, respectively. The nitrite/nitrate content of haemocytes and lymph were found to be 5 and 50 μM, respectively. The detected nitrite reduction activity of haemocytes allowed us to estimate the generation rate of nitric oxide as 0.05-0.5 nM/min from endogenous nitrite. It is thus assumed that the observed nitrite reduction activity in haemocytes is dominant in the increased NO production during immune response of the G. mellonella larvae.

Effect of selenolipoic acid on peroxynitrite-dependent inactivation of NADPH-cytochrome P450 reductase

Seleno-organic compounds are known as efficient “scavengers” of peroxynitrite (PN). Here we studied the protective effect of selenolipoic acid (SeLA), the seleno-containing analogue of lipoic acid, on peroxynitrite-dependent inactivation of NADPH-cytochrome P450 reductase. 3-Morpholinosydnonimine hydrochloride (SIN-1) was used as a source of peroxynitrite. The reductase was irreversibly inactivated by PN generated from SIN-1. The inactivation occurred with the rate constant of about 3 × 104M-1s-1. The presence of SeLA at low concentration (0.5 μM) led to synergistic increase of the reductase inactivation by PN. Our results suggest the formation of a reactive derivative of SeLA in the reaction of SeLA with PN, probably selenolseleninate, that mediates the aggravation of reductase inactivation. In the presence of SeLA, the inactivation was reversible under the action of thiols, allowing us to conclude that the observed action of SeLA may be considered as protective.

VENOM FROM THE ECTOPARASITIC WASP Habrobracon hebetor ACTIVATES CALCIUM-DEPENDENT DEGRADATION OF Galleria mellonella LARVAL HEMOCYTES.

Ectoparasitoids inject venom into hemolymph during oviposition. We determined the influence of envenomation by the parasitoid, Habrobracon hebetor, on the hemocytes of its larval host, Galleria mellonella. An increase in both intracellular Са(2+) content and phospholipase C activity of the host hemocytes was recorded during 2 days following envenomation by the parasitoid. The decreased hemocyte viability was detected 1, 2, and 24 h after the envenomation. Injecting of the crude venom (final protein concentration 3 μg/ml) into the G. mellonella larvae led to the reduced hemocyte adhesion. The larval envenomation caused a decrease in transmembrane potential of the hemocytes. These findings document the suppression of hemocytic immune effectors in the parasitized host larvae.

Detection of free radicals formation in haemolymph of insects by EPR spectroscopy

The generation of reactive oxygen species during oxidation of DOPA (3-(3,4-dihydroxyphenyl-DL-alanine) in haemolymph of insects has been studied by electron paramagnetic resonance with spin traps 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and 1-hydroxy-3-carboxy-pyrrolidine. The data support the formation of DOPA-derived highly reactive intermediates during melanization in haemolymph of insects. The formation of superoxide has not been detected apparently due to reaction of DOPA with superoxide. The rate constant of the latter reaction was estimated ask≈5·105 M−1.c−1. The formation of DOPA-semiquinone in haemolymph has been measured using spin stabilization ofo-semiquinones by Mg2+. The comparative studies in haemolymph of intact insects and insects infected by fungal infection suggest an important role of DOPA-semiquinone production in the immune status of insects.

Affinity modification of NADPH-cytochrome P-450 reductase

The active centre of NADPH-cytochrome P-450 reductase contains the lysine residue essential for catalytic activity. Chemical modification of epsilon-amino group of this lysine residue is the subject of the present study. To modify the epsilon-amino group, we have employed the periodate-oxidized NADP+ and NAD+ (o-NAD(P]. The both reagents have appeared to be the competitive inhibitors of NADPH-cytochrome P-450 reductase (Ki for o-NADP approximately 10 microM, Ki for o-NAD greater than 100 microM). However, o-NADP has not a covalency bond with reductase, whilst o-NAD modifies the reductase at the binding site of NADPH. A protective effect of NADP+ and the labeling extent close to unity (0.7) at deep reductase inactivation indicate the affinity type of this modification. Different results of reductase modification by either o-NADP or o-NAD may be due to the difference in the structures of the analogs bound to the enzyme active site.

Dual effect of nitric oxide on phenoloxidase-mediated melanization

Abstract The study has demonstrated a dual effect of nitric oxide on phenoloxidase (PO)-mediated DOPA oxidation and melanization process. NO generated at low rates proportionally increased in PO-mediated DOPA oxidation. Competitive PO inhibitor, phenylthiourea, resulted in significant inhibition of NO-mediated DOPA oxidation. Further analysis using fluorescent and EPR methods demonstrated that the effect of NO on DOPA oxidation is explained by oxidation of NO to NO2 at the active site of PO followed by oxidation of DOPA by NO2. On the contrary, the bolus addition of NO gas solution resulted in a significant decrease in observed PO activity. Similar dose-dependent effect of NO was observed for the insect’s haemocytes quantified as percentage of melanized cells after treatment with nitric oxide. In conclusion, the results of the study suggest that NO may have a significant regulatory role on melanization process in invertebrates as well as in human and result in protective or damaging effects.

Light-Stimulated Generation of Free Radicals by Quinones-Chelators

Abstract The role of metal ions in the mechanism of light-stimulated redox activity of potential anticancer agent 2-phenyl-4-(butylamino)naphtha[2,3-h]quinoline-7,12-dione (Qc) has been studied by CIDNP (chemically induced dynamic nuclear polarization) and EPR methods. The photo-induced oxidation of NADH and its synthetic analog – substituted dihydropyridine (DHP) – by quinone Qc was used as a model. The Qc capability of producing chelating complexes with divalent metal ions of Fe, Zn and Ca was studied quantitatively by optical absorption spectroscopy. A significant decrease of electrochemical reduction potential of Qc (ΔE=0.4−0.6 eV for ACN and ACN/PBS solutions) in chelating complexes and in protonated form of Qc was observed. A pronounced increase in efficiency of DHP oxidation in chelating complexes with Zn2+ and Ca2+ ions compared with free Qc was demonstrated. The yields of free radicals, including reactive oxygen species (ROS) and reaction products, were a few times higher than those in the absence of metal ions. Application of such chelating compounds to enhance ROS generation looks very promising for anti-cancer therapy, including the photodynamic therapy.