V. S. Bondar

Sequence of the cDNA encoding the Ca2+-activated photoprotein obelin from the hydroid polyp Obelia longissima ☆

A cDNA clone encoding the Ca(2+)-activated photoprotein, obelin (Obl), from Obelia longissima was sequenced. The nucleotide (nt) sequence contained two long overlapping open reading frames (ORFs), one of which encoded apoobelin (apoObl). The deduced amino acid (aa) sequence of apoObl revealed that this 195-aa protein has three EF-hand structures that are characteristic for Ca(2+)-binding domains. Strong aa homology was shown among apoObl, apoaequorin and apoclytin. The second ORF present in the obl cDNA consists of 139 codons and encodes a very basic protein with a calculated pI of 10.56 and a molecular mass of 16,153 Da.

19F NMR study on the biodegradation of fluorophenols by various Rhodococcus species

Of all NMR observable isotopes 19F is the one perhaps most convenient for studies on biodegradation of environmental pollutants. The reasons underlying this potential of 19F NMR are discussed and illustrated on the basis of a study on the biodegradation of fluorophenols by four Rhodococcus strains. The results indicate marked differences between the biodegradation pathways of fluorophenols among the various Rhodococcus species. This holds not only for the level and nature of the fluorinated biodegradation pathway intermediates that accumulate, but also for the regioselectivity of the initial hydroxylation step. Several of the Rhodococcus species contain a phenol hydroxylase that catalyses the oxidative defluorination of ortho-fluorinated di- and trifluorophenols. Furthermore, it is illustrated how the 19F NMR technique can be used as a tool in the process of identification of an accumulated unknown metabolite, in this case most likely 5-fluoromaleylacetate. Altogether, the 19F NMR technique proved valid to obtain detailed information on the microbial biodegradation pathways of fluorinated organics, but also to provide information on the specificity of enzymes generally considered unstable and, for this reason, not much studied so far.

The Chemical Basis of Fungal Bioluminescence

Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

Size effect on the optical limiting in suspensions of detonation nanodiamond clusters

We report on the optical limiting (OL) in stable aqueous suspensions of detonation nanodiamond (ND) clusters with average size of 50, 110, and 320 nm. The nanosecond Z-scan measurements at wavelength of 532 nm revealed that the larger the cluster size, the better the OL performance and the higher the ray stability of the ND suspension. Our analysis showed that the nonlinear scattering and the nonlinear absorption are the dominant mechanisms of OL in aqueous ND suspensions.

Nonlinear scattering of light in nanodiamond hydrosol

The nonlinear scattering of light under the conditions of optical limiting of nanosecond pulsed laser radiation at a wavelength of 1064 nm in a nanodiamond (ND) hydrosol has been experimentally studied. Superstable hydrosols were obtained from detonation NDs with a modified surface. Using an improved scheme of z scanning, it is shown that a decrease in the optical transmission coefficient of an ND hydrosol under optical limiting conditions is due to enhanced nonlinear scattering. It is established that the energy of pulsed radiation scattered at a right angle obeys a power law in dependence on the energy density of incident radiation pulses. Hydrosols of detonation NDs with the modified surface exhibit high stability with respect to the periodic laser action at high power density.

ATP is a cosubstrate of the luciferase of the earthworm Fridericia heliota (Annelida: Clitellata: Oligochaeta: Enchytraeidae).

The luminescence system of the soil enchytreid Fridericia heliota (this species has been discovered in 1990 [1]) is peroxide-independent and belongs to the luciferin–luciferase type [2]. Certain general properties of this luminescence system were studied both in vivo and in vitro. It was found that the reaction of light emission includes at least four basic components: an enzyme (luciferase), a reaction substrate (luciferin), magnesium ions, and oxygen [2]. According to preliminary data, the native molecular weight of the F. heliota luciferase is about 40 kDa. Because the activity of the enzyme rapidly declines during purification, the pure preparation of the F. heliota luciferase has not yet been obtained. Although the thermal stability of the luciferase preparation obtained by alkaline (or acid) extraction from worms was high, the ability of the resultant preparation to initiate luminescence was lost during long-term storage. The goal of this work was to study the ATP-dependent luminescence of F. heliota and to elucidate specific properties of the reaction substrate. It was shown in the preceding work [2] that the addition of adenosine phosphates (ATP, ADP, and AMP) to reaction mixture containing the preparations of luciferase and luciferin caused inhibition of luminescence. It was suggested that the effect of inhibition observed in these experiments was due to chelation of magnesium ions by phosphate groups, because magnesium ions contained in preparations were involved in the reaction of light emission. A detailed study of this effect gave rise to a completely new insight into the luminescence system of F. heliota. The final concentration of adenosine phosphates in reaction mixture was more than 30 times higher than the concentrations of magnesium ions the (concentration of magnesium ions in preparations of luciferase and luciferin, determined by the method of atomic absorption analysis, were similar and equaled to approximately 2.8 × 10 –4 M). The addition of an excess of magnesium ions should restore the luminescence intensity to the initial level. However, it was found that the intensity of the light emission was not only restored to the initial level but even exceeded this level by many times (Fig. 1, curve a ). A repeated addition of ATP caused no changes in the luminescence kinetics, which was indicative of saturating concentration of ATP in reaction mixture. Therefore, the decrease in the luminescence intensity was caused by other factors.

Study of the luminescence system of the soil enchytraeid Fridericia heliota (Annelida: Clitellata: Oligochaeta: Enchytraeidae).

Luminescent earthworms were first observed in Russia almost 200 years ago. In 1838, luminescent oligochaetes, identified as a new species Lumbricus noctilicus , were found in Kazan [1]. They emitted weak luminescence distributed over the body, with a greater intensity at the ends, and left luminescent mucus on the fingers of the observer. Later, upon more thorough studying, these worms were classified with the earlier described species Enchytraeus albidus (Henle, 1837) [2]. In 1908, the occurrence of luminescent soil oligochaets in the Kaluga guberniya (region) and in Perm’ were reported. The worms discovered were identified as Henlea ventriculosa . It is interesting that, in the same work, the name Enchytraeus albidus was used as a synonym of Henlea ventriculosa [3]. The luminescence of these oligochaetes was their intrinsic property, not related to a possible contamination with luminescent bacteria [4]. Thereafter, for almost 80 years, luminescent earthworms were not observed in Russia.

Cadmium-induced luminescence of recombinant photoprotein obelin.

Abstract It has been shown for the first time that Cd2+ ions induce substantial bioluminescence of a Ca2+-binding photoprotein: recombinant obelin. The optimum pH for the bioluminescent reaction in the presence of Cd2+ ions is pH 6. The intensity, L, of the light emission for the Cd2+ ions is 75% of the intensity of the signal in the presence of Ca2+. The quantum yields of the reactions in the presence of Cd2+ and Ca2+ are 0.18 and 0.24 respectively. The slope of the straight line (between 5 and 90% of Lmax in the coordinates of log( L (L max -L) ) vs. log([Cd2+]) is 1.75 ± 0.06, which indicates positive cooperative character of this reaction. At a concentration exceeding 1 · 10−3 M, Cd2+ inhibits the bioluminescent reaction.

Isolation of luminescence system from the luminescent fungus Neonothopanus nambi

56 This study is devoted to the problem of isolation of the lighttemitting system able to luminesce in vitro from the fungus Neonothopanus nimbi. The study was performed with the mycelium of the luminous higher fungus N. nambi, inhabiting the tropp ical forests of South Vietnam. [1] The fungus culture was kindly provided for experiments by Vietnamese researcher Dao Thi Van (private collection of strains To obtain fungal biomass, the mycelium was cultured in Petri plates in a liquid nutrient medium by the techh nology described earlier [2]. The grown mycelium was taken from the Petri dish and washed with deionized (DI) water (MilliiQ system, Millipore, United States) to remove the residual components of the nutrient medium and exometaboliltes. After washing, the remaining water was removed from the mycelial biom ass with filter paper. The isolation of the luminescent system of N. nambi mycelium included the following steps, which were carried out at 0–4°C. Mycelium washed with DI water was ground in the cold with scissors and transferred to a beaker placed in an ice bath. The bioo mass was poured with cold 0.1 M phosphate buffer (pH 7.0) supplemented with 0.1–1.0% BSA (Serva, Germany) in the ratio 1 : 5 (wet biomass weight : buffer volume). The biomass was destroyed with a Volna ultrasonic disintegrator (Russia). Sonication was perr formed at a power of 200 W three times for 5 s at 11min intervals. The homogenate was transferred into chilled tubes and centrifuged at 48 000 g for 30–60 min in an Avanti ® JE centrifuge (BeckmannCoulter, United States). The pellet was discarded, and the supernatant was either used immediately for study (in this case, it was stored at 4°C) or immediately frozen at –20°C and stored at this temperature. The luminescence of the supernatants was meaa sured using a BLM 8801 luminometer (Nauka Special Engineering and Design Department, Krasnoyarsk, Russia) calibrated using the Hastings–Weber radioacc tive standard [3] (one luminescent unit was 10 8 phoo tons per 1 s). The signals were recorded using an LKB 2210 recorder (LKB, Sweden). It was found that supernatants isolated from the mycelium of the luminous fungus N. nambi by the method described above emitted long luminescence (Fig. 1). This fact allowed us to conclude that a selff sufficient luminescent system that ensures luminess cence in vitro was isolated from this fungal species. After filtering the supernatant through a membrane with an …

Chemiluminescent emission of tissues of fruit bodies of higher fungi

105 Among the dozens of thousands of species of higher fungi known to date, more than 80 species possess bioluminescence—the ability to emit light that is visii ble with the naked eye. This ability was found in the to note that the luminescent species phylogenetically coexist with the nonluminescent ones. Sometimes even one taxonomically defined species includes both luminescent and nonluminescent forms [2, 3]. Such a mosaic distribution of bioluminescence suggests that the ability to luminesce has occurred in the kingdom of fungi repeatedly and independently and that its evolutionary basis is a fundamental bioo chemical process, a small deviation in which (even in one or two stages of the metabolic chain) gives rise to luminescence. However, such a process remains unree vealed as yet. The absence of intermediate forms makes it highly difficult to elucidate the evolutionary pathway of the emergence of bioluminescence in the kingdom of fungi. Although the notions on the mechanism of light emission by fungi are far from complete [3, 4], it is obviously distinct from the understood emission mechanisms in animals and bacteria. Weak chemiluu minescence is characteristic of animal tissues [5]. It is known that the major source of luminescence in animals is lipid peroxidation [6] and that chemilumii nescence in plants is related to the photosynthesis system [7]. We studied the emission of nonbioluminescent higher fungi. Studies were performed with different species of higher fungi growing in forests of the Eastt ern Siberian region of Russia (Krasnoyarsk Krai). The objects of the study were 150 samples of fungi collected in forests in vicinities of Krasnoyarsk in summer 2011. The collections were representatives of five orders, 15 families, 21 genera, and 13 species. As many as 136 samples of the collected material were identified to the genus level and 35 samples to the species level (table). We measured luminescence of fungal fragments taken from different parts of the fruit body. Measuree ments were performed with the Glomax 20/20 lumii nometer (Promega, United States), which was calii brated using the Hastings–Weber radioactive standard [8] (2.7 × 10 3 quanta/s was taken as one unit of lumii nescence). The emission of each sample was recorded for 10 s. Signals exceeding the background level by at least 5 times were taken as reliable. Then, each sample was air dried to a constant weight to calculate the spee cific luminosity per unit mass. The table shows the …