ISSN 16076729, Doklady Biochemistry and Biophysics, 2013, Vol. 449, pp. 80–83. © Pleiades Publishing, Ltd., 2013. Original Russian Text © V.S. Bondar, E.K. Rodicheva, S.E. Medvedeva, N.A. Tyulkova, A.B. Tyaglik, B.A. Shpak, J.I. Gitelson, 2013, published in Doklady Akademii Nauk, 2013, Vol. 449, No. 2, pp. 223–227. 80 Several tens of higher fungal species displaying bioluminescence, the ability to emit visible light, have been described so far [1, 2]. However, the nature of this phenomenon in the kingdom of fungi still remains unclear unlike the luminescence of bacteria and ani mals. The mechanism underlying luminescence has been studied for many animal and bacterial species and even formed the background for a new branch of biochemical analytics, which has found a wide appli cation in biology and medicine. The bioluminescence of fungi has not been used in this field, since the molecular mechanism of their light emission is rather vague. There are two alternative standpoints on the mechanism of fungal bioluminescence: (i) their biolu minescence is associated with the classic enzyme– substrate system luciferase–luciferin, analogous to that of bacteria and animals [3, 4], and (ii) it is pro duced via a chemiluminescence reaction without involvement of a specific enzyme [5, 6]. We discovered and measured chemiluminescence in many species of “nonluminescent” higher fungi [7]. Shimomura [8] earlier described chemiluminescence of a nonluminescent strain of the luminescent fungus Panellus stipticus. Mihail and Bruhn [9] discovered luminescence of hyphae in 13 fungal species belonging to the phyla Basidiomycota, Ascomycota, and Zygo mycota. Together, these data give grounds to assume that chemiluminescence in general is characteristic of the metabolism of higher fungi and suggest that fungal chemiluminescence was the metabolic basis for emer gence of visible fungal bioluminescence during evolu tion via intensification of function. To test these hypotheses, we studied the specific luminescence features of the higher fungus Neonotho panus nambi, inhabiting tropical forests of South Viet nam, described, and maintained in culture [10]. The experiments were conducted with N. nambi mycelium specimens obtained by submerged cultiva tion in a liquid potato–sucrose nutrient medium (200 g potato, 20 g sucrose, and 1 L distilled water) in 500mL flasks containing 100 mL of the medium. Sus pension of minced mycelium grown in petri dishes according to the earlier developed technology [11] was used as an inoculum. The inoculum volume was 5– 10% of the nutrient medium. The fungus was culti vated for 3 days at a temperature of 28°C and constant stirring at 200 rpm in an ES20 (BIOSAN, Latvia) environmental shakerincubator. The luminescence of mycelium was recorded in a BLM 8801 (Design and Engineering Office SKTB Nauka, Krasnoyarsk, Russia) luminometer calibrated according to the Hastings–Weber radioactive standard [12] (one luminescence unit is 10 8 photons/s). Myce lium specimens were placed into a luminometer cuvette with 500 μL of deionized (DI) water (MilliQ system, Millipore, United States) to record the light signals with a 2210 (LKB, Sweden) selfrecorder. Spectral analysis of N. nambi mycelium extracts was carried out using a Uvikon 943 UV/VIS spectro photometer (Kontron Instruments, Italy) and a Varian Cary Eclipse spectrofluorometer (Agilent Technolo gies, United States). The extracts were produced by mechanical disintegration of mycelium in DI water using a glass–glass homogenizer with subsequent removal of the cell debris by centrifugation at 16 000g (5415R centrifuge, Eppendorf, Germany) for 15 min at 10°C. Our technology for submerged cultivation allows for growing N. nambi spherical mycelial globules (Fig. 1) with a pronouncedly rough surface formed by numer ous projections. The diameter of the globules grown using the selected cultivation method and growth con ditions varied in the range of 2–7 mm. Globular myce lium has certain advantages for examination. The globules are easily transferred from one liquid medium to another and readily fit into the luminometer cuvette with a spatula with minimal mechanical impact on the fungus and without injuries. It has been shown that the mycelial globules trans ferred to a measuring cuvette from the nutrient medium display no luminescence; that is, the lumi On the Mechanism of Luminescence of the Fungus Neonothopanus nambi V. S. Bondar a, b , E. K. Rodicheva a, b , S. E. Medvedeva a, b , N. A. Tyulkova a, b , A. B. Tyaglik b , B. A. Shpak b , and Academician J. I. Gitelson a, b Received July 16, 2012 DOI: 10.1134/S1607672913020075 a Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk, Russia b Siberian Federal University, Krasnoyarsk, Russia BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY