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.     

Express Method for Determination of Low Value of Trans-membrane Potential of Living Cells with Fluorescence Probe: Application on Haemocytes at Immune Responses

The method for measurement of trans-membrane potential of cell membrane was evaluated for the case of low potential value using fluorescence probe 4-(4-dimethylaminostyryl)-1-methylpyridinium, DSM. The method is based on comparative titration of cells with probe in buffers containing Na(+) or K(+). The apparent trans-membrane potential obtained with this way is a result of K(+)-Na(+) pump activity. The presented approach allowed measuring the low value of potential with 1-2?mV of accuracy without additional calibration procedures. The method was applied for investigation of potential of cell membrane of haemocytes of Galleria mellonella larvae. The value of potential of intact insect's haemocytes was found in the range from -10 to -20?mV. The change of potential value of haemocytes was investigated under model immune response and natural envenomation and parasitizing. The obtained deviations of cell membrane potential were in good correlation with changes of activity of main immune reactions, described in literature and obtained by us earlier.     

Pathogen-targeted hydroxyl radical generation during melanization in insect hemolymph: EPR study of a probable cytotoxicity mechanism

The main component of the insect immune system is melanotic encapsulation of pathogenic organisms. Molecular mechanisms of destruction of an encapsulated pathogen are poorly understood. Reactive oxygen species (ROS) are considered as probable cytotoxic agents responsible for destruction of pathogenic organisms in insect hemolymph. In the present work the formation of H₂O₂ during melanization in Galleria mellonella hemolymph in the presence of catalase inhibitor NaN₃ was detected. Enhanced rates of H₂O₂ generation were observed in the hemolymph of insects activated by injection of bacterial cells. Using spin trapping technique in combination with electron paramagnetic resonance spectroscopy we demonstrated that production of H₂O₂ in the hemolymph causes the formation of highly toxic reactive oxygen species, hydroxyl radical. However, neither H₂O₂ nor hydroxyl radical were detected in the absence of NaN₃ in agreement with the high catalase activity in the hemolymph. These observations allow us to propose a unique mechanism of pathogen-targeted cytotoxicity based on localized hydroxyl radical generation within a melanotic capsule.