Finding the Light Emission Stimulator of Neonothopanus nambi Basidiomycete and Studying Its Properties.

A stimulator of light emission of the fungus was found in an aqueous extract from mycelium of the luminous basidiomycete Neonothopanus nambi after its treatment with ß-glucosidase. The addition of the extract to the luminous mycelium increases the level of light emission from several times to 1.5 orders of magnitude or more. The luminescence stimulator is a low-molecular-weight thermostable compound: it is detected in the permeate after filtering the extract through a 10-kDa cutoff membrane and it retains the stimulating effect after heat treatment at 100°C for 5 min. In the absorption spectrum of the aqueous sample of the stimulator, two main peaks are observed in the shortwave region (205 and 260 nm) and a shoulder in the range of 350-370 nm can be seen. The luminescence stimulator exhibits blue fluorescence with an emission maximum at 440 nm when excited at 360 nm. It was established that the luminescence-stimulating component is not a substrate (or its precursor) of the luminescent system of the N. nambi fungus.

Reusable System for Phenol Detection in an Aqueous Medium Based on Nanodiamonds and Extracellular Oxidase from Basidiomycete Neonothopanus nambi.

A reusable system for phenol determination in an aqueous medium was obtained by adsorption of extracellular oxidase from fungus Neonothopanus nambi onto modified nanodiamonds (MND) synthesized by detonation. It was found that the enzyme strongly binds to MND and exhibits catalytic activity in the reaction of co-oxidation of phenol with 4-aminoantipyrine without the addition of hydrogen peroxide. In the presence of the MND-oxidase complex, a significantly (by an order of magnitude) higher yield of the reaction product is recorded as compared to the yield in the presence of a free enzyme; the mechanism of the revealed effect is discussed. Model experiments have demonstrated the multiple use of the MND-oxidase complex for testing phenol in aqueous samples. The immobilized enzyme exhibits functional activity during long-term (2 months) storage of the MND-oxidase complex at 4°C. The data obtained create the prerequisites for using the created system in environmental monitoring of water pollution with phenol.

Extracellular Oxidases of Basidiomycete Neonothopanus nambi: Isolation and Some Properties.

Using the original technique of treating biomass with ß-glucosidase, a pool of extracellular fungal enzymes was obtained for the first time from the mycelium of basidiomycete Neonothopanus nambi. Two protein fractions containing enzymes with oxidase activity were isolated from the extract by gel-filtration chromatography and conventionally called F1 and F2. Enzyme F1 has a native molecular weight of 80-85 kDa and does not contain chromophore components; however, it catalyzes the oxidation of veratryl alcohol with Km = 0.52 mM. Probably, this enzyme is an alcohol oxidase. Enzyme F2 with a native molecular weight of approximately 60 kDa is a FAD-containing protein. It catalyzes the cooxidation of phenol with 4-aminoantipyrine without the addition of exogenous hydrogen peroxide, which distinguishes it from the known peroxidases. It was assumed that this enzyme may be a mixed-function oxidase. F2 oxidase has Km value 0.27 mM for phenol. The temperature optimums for oxidases F1 and F2 are 22-35 and 55-70°C, and pH optimums are 6 and 5, respectively.

Creation of Bifunctional Indicating Complex Based on Nanodiamonds and Extracellular Oxidases of Luminous Fungus Neonothopanus nambi.

A bifunctional indicating complex was created by immobilization of extracellular oxidases (glucose oxidase and peroxidases) of luminous fungus Neonothopanus nambi onto modified nanodiamonds (MNDs) synthesized by detonation. It was found that the enzymes firmly adsorb onto MND particles and exhibit their catalytic activity. Model in vitro experiments showed that the created MND-enzymes complex is suitable for repeated use for analyte (glucose and phenol) testing and retains its activity after storage at 4°C in deionized water for 1 month. The data obtained offer the prospects for developing a new class of reusable multifunctional indicating and diagnostic test systems on the basis of MNDs and higher fungal enzymes for medical and ecological analytics.

Effect of tritium on luminous marine bacteria and enzyme reactions.

The paper studies chronic effect of tritiated water, HTO, (0.0002-200 MBq/L) on bioluminescent assay systems: marine bacteria Photobacterium phosphoreum (intact and lyophilized) and coupled enzyme reactions. Bioluminescence intensity serves as a marker of physiological activity. Linear dependencies of bioluminescent intensity on exposure time or radioactivity were not revealed. Three successive stages in bacterial bioluminescence response to HTO were found: (1) absence of the effect, (2) activation, and (3) inhibition. They were interpreted in terms of reaction of organisms to stress-factor i.e. stress recognition, adaptive response/syndrome, and suppression of physiological function. In enzyme system, in contrast, the kinetic stages mentioned above were not revealed, but the dependence of bioluminescence intensity on HTO specific radioactivity was found. Damage of bacteria cells in HTO (100 MBq/L) was visualized by electron microscopy. Time of bioluminescence inhibition is suggested as a parameter to evaluate the bacterial sensitivity to ionizing radiation.

Bioluminescence as a tool for studying detoxification processes in metal salt solutions involving humic substances.

The paper considers effects of humic substances (HS), as natural attenuators of toxicity, on solutions of model inorganic pollutants, metal salts - Pb(NO(3))(2), СоСl(2), CuSO(4), Eu(NO(3))(3), СrСl(3), and K(3)[Fe(СN)(6)]. Luminous bacteria Photobacterium phosphoreum and bioluminescent system of coupled enzymatic reactions were used as bioassays to monitor toxicity of salt solutions. The ability of HS to decrease or increase toxicity was demonstrated. Detoxifying concentrations of HS were determined; detoxification coefficients were calculated at different times of exposure of salt solutions to HS. To study the combined effects of HS and salts on bioluminescent assay systems, the rates of biochemical reactions and bacterial ultrastructure were analyzed. The detoxifying effects were explained by: (1) decrease of free metal content in water solutions under metal-HS binding; (2) increase of biochemical reaction rates in a bioluminescent assay system under HS effect; (3) enhancement of mucous layers on cell surface as a response to unfavorable impact of toxicants. Detoxifying mechanisms (2) and (3) reveal the active role of bioassay systems in detoxification processes.

Tissue reaction to intramuscular injection of resorbable polymer microparticles.

Tissue reaction to implantation of polymeric microparticles from resorbable polymer (polyhydroxybutyrate) is characterized by slight inflammatory reaction and pronounced progressive macrophage infiltration with the presence of mono- and multinuclear foreign body giant cells resorbing the polymeric matrix. No fibrous capsules were formed around the polymeric microparticles; neither necrosis nor other adverse morphological changes and tissue transformation in response to implantation of the PHB microparticles were recorded. The results indicate good prospects of using polyhydroxybutyrate for the construction of long-acting dosage forms as microparticles for intramuscular injection.

Population dynamics of transgenic strain Escherichia coli Z905/pPHL7 in freshwater and saline lake water microcosms with differing microbial community structures.

Populations of Escherichia coli Z905/pPHL7, a transgenic microorganism, were heterogenic in the expression of plasmid genes when adapting to the conditions of water microcosms of various mineralization levels and structure of microbial community. This TM has formed two subpopulations (ampicillin-resistant and ampicillin-sensitive) in every microcosm. Irrespective of mineralization level of a microcosm, when E. coli Z905/pPHL7 alone was introduced, the ampicillin-resistant subpopulation prevailed, while introduction of the TM together with indigenous bacteria led to the dominance of the ampicillin-sensitive subpopulation. A high level of lux gene expression maintained longer in the freshwater microcosms than in sterile saline lake water microcosms. A horizontal gene transfer has been revealed between the jointly introduced TM and Micrococcus sp. 9/pSH1 in microcosms with the Lake Shira sterile water.

Biofilm formation by bacterial associations under various salinities and copper ion stress.

The study addresses the effect of abiotic (medium salinity and copper ions) and biotic (interactions between populations) factors on the formation of structured communities by binary associations consisting of halotolerant bacteria (Alcaligenes sp. 1-1 or Acinetobacter sp. 1-19) and a wild-type B. subtilis 2335 strain or a transgenic strain. The results showed that 250 mg l(-1) of copper ions inhibit formation of biofilms by monocultures of the tested strains. Binary associations of the strains were more resistant to high concentrations (250 mg l(-1)) of copper ions. At the lowest NaCl concentration (0.05% and 2.5%) and in the presence of copper ions, bacilli seemed to help halotolerant bacteria survive. Under increased salinity and in the presence of copper ions, structured communities developed due to halotolerant bacteria. Coexistence under stressful conditions was beneficial for the both groups of bacteria.

Tissue response to the implantation of biodegradable polyhydroxyalkanoate sutures.

Polyhydroxyalkanoate (PHA) sutures were implanted to test animals intramuscularly, and tissue reaction was investigated and compared with the reaction to silk and catgut. Tested monofilament sutures made of PHAs of two types--polyhydroxybutyrate (PHB) and a copolymer of hydroxybutyrate and hydroxyvalerate (PHV)--featured the strength necessary for the healing of muscle-fascial wounds. The reaction of tissues to polymeric implants was similar to their reaction to silk and was less pronounced than the reaction to catgut; it was expressed in a transient post-traumatic inflammation (up to four weeks) and the formation of a fibrous capsule less than 200 microm thick, which became as thin as 40-60 microm after 16 weeks, in the course of reverse development. Macrophages and foreign-body giant cells with a high activity of acid phosphatase were actively involved in this process. PHB and PHB/PHV sutures implanted intramuscularly for an extended period (up to one year) did not cause any acute vascular reaction at the site of implantation or any adverse events, such as suppurative inflammation, necrosis, calcification of the fibrous capsule or malignant tumor formation. No statistically significant differences were revealed in the tissue response to polymer sutures of the two types. Capsules around silk and catgut sutures did not become significantly thinner.

Immunoelectronmicroscopic study of the nucleoid structure of hydrogen bacteria.

Electron microscopical studies of the nucleoid structure of hydrogen bacteria using ultrahin sections and spread DNA from bacterial cell lysates revealed a different DNA packaging in the cell. A compact state of the major part of DNA at all growth stages and stability of nucleosome-like structures were shown. The use of antibodies to HU protein of E. coli labelled by protein A-colloidal gold demonstrated the immunological relationship between HU protein of E. coli and histone-like proteins of Alcaligenes eutrophus and their possible role in the nucleosome-like DNA packaging in procariotic genome.