[Laparoscopic management of the prostatic utricle cyst in a 4-year-old child].

The article present the clinical observation of 4 year old boy with scrotal hypospadias. Previous surgery management had unsatisfactory results due to complications of the cyst of the prostatic utricle. Laparoscopic removal of the cyst of the prostatic utricle was performed. The next step was urethral plastic. The results of the operation were satisfactory. A brief review of the literature is provided.

Structural study of the X-ray-induced enzymatic reduction of molecular oxygen to water by Steccherinum murashkinskyi laccase: insights into the reaction mechanism.

The laccase from Steccherinum murashkinskyi is a member of the large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates, accompanied by the reduction of dioxygen to water. The reducing properties of X-ray radiation and the high quality of the laccase crystals allow the study of the catalytic reduction of dioxygen to water directly in a crystal. A series of diffraction data sets with increasing absorbed radiation dose were collected from a single crystal of Steccherinum murashkinskyi laccase at 1.35 Šresolution. Changes in the active-site structure associated with the reduction of molecular oxygen to water on increasing the absorbed dose of ionizing radiation were detected. The structures in the series are mixtures of different states of the enzyme-substrate complex. Nevertheless, it was possible to interpret these structures as complexes of various oxygen ligands with copper ions in different oxidation states. The results allowed the mechanism of oxygen reduction catalyzed by laccases to be refined.

Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

Dipeptide carnosine (ß-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.

Biocatalytic conversion of poultry processing leftovers: Optimization of hydrolytic conditions and peptide hydrolysate characterization.

Peptide hydrolysate (PH) was produced by deep controllable bioconversion of poultry processing leftovers (broiler necks), by means of a multienzyme composition, containing four commercially available enzyme preparations (Alcalase, Neutrase, Flavourzyme, Protamex). The design of multienzyme composition (MEC) was applied to yield a hydrolysate with adjusted properties, including minimized antigenicity and bitterness. The protein recovery was optimized using Box-Behnken response surface design. The individual and interactive effects of hydrolysis conditions (time, hydromodule and MEC dosage) were studied. The experimental data were analyzed by ANOVA method and a well-predictive, second order polynomial model was developed using multiple regression analysis. Optimal hydrolysis conditions were found to be: hydrolysis time 3 h, hydromodule 2.25 l/kg and dosage of MEC 0.25%. The corresponding predicted value for protein recovery was 75.34%, 2 times higher compared to traditional long-term heating hydrolysis. The PH obtained is a low allergenic product with high antioxidant capacity.

[Preparation of protoplasts of the fungus Trametes hirsuta 072 and study of the effect of antioxidants on their formation and regeneration].

The consistent application of homogenization and enzymatic treatment is required to obtain protoplasts from the basidiomycete fungus Trametes hirsuta. The maximum yield of protoplasts (∼2.5 × 107/mL) was achieved when mycelium in the exponential growth phase (60 h) was used. The maximum stability was observed in MES+ buffer during 4 h of incubation; in this case the titer reduction was 5­7%. Studies of the effect of antioxidants with different antioxidant capacities expressed in mmol equivalents of Trolox (ascorbate, 0.99; α-tocopherol, 1.0; ß-carotene, 2.14; quercetin, 3.98) indicated that the yield of protoplasts was increased in the presence of ß-carotene and quercetin by 18­24%. The studied antioxidants did not affect the protoplasts stability. The degree of regeneration of protoplasts correlated with the antioxidant capacity of the studied antioxidants and was maximal (0.4%) in the presence of ß-carotene and quercetin; it was 0.1% in the presence of MES+. The rate of protoplast growth was two times higher in the presence of ß-carotene and quercetin.

[Degradation of fluorene and fluoranthene by the basidiomycete Pleurotus ostreatus].

The dependence of the degree of fluorene and fluoranthene degradation by the fungus Pleurotus ostreatus D1 on the culture medium composition has been studied. Polycyclic aromatic hydrocarbons (PAHs) have been transformed in Kirk's medium (under conditions of laccase production) with the formation of a quinone metabolite and 9-fluorenone upon the use of fluoranthene and fluorene as substrates, respectively. More complete degradation with the formation of an intermediate metabolite, phthalic acid that has undergone subsequent utilization, has occurred in basidiomycete-rich medium (under the production of both laccase and versatile peroxidase). The formation of phthalic acid as a metabolite of fluoranthene degradation by lignolytic fungi has been revealed for the first time. The data allow the supposition that both extracellular laccase and laccase on the mycelium surface can participate in the initial stages of PAH metabolism, while versatile peroxidase is necessary for the oxidation of the formed metabolites. A scheme of fluorene metabolism by Pleurotus ostreatus D1 is suggested.

[Oil degradation by basidiomycetes in soil and peat at low temperatures].

A total of 17 basidiomycete strains causing white rot and growing on oil-contaminated substrates have been screened. Three strains with high (Steccherinum murashkinskyi), average (Trametes maxima), and low (Pleurotus ostreatus) capacities for the colonization of oil-contaminated substrates have been selected. The potential for degrading crude oil hydrocarbons has been assessed with the use of fungi grown on nonsterile soil and peat at low temperatures. Candida sp. and Rhodococcus sp. commercial strains have been used as reference organisms with oil-degrading ability. All microorganisms introduced in oil-contaminated soil have proved to be ineffective, whereas the inoculation of peat with basidiomycetes and oil-degrading microorganisms accelerated the destruction of oil hydrocarbons. The greatest degradation potential of oil-aliphatic hydrocarbons has been found in S. murashlinskyi. T. maxima turned out to be the most successful in degrading aromatic hydrocarbons. It has been suggested that aboriginal microflora contributes importantly to the effectiveness of oil-destructing microorganisms. T. maxima and S. murashkinskyi strains are promising for further study as oil-oxidizing agents during bioremediation of oil-contaminated peat soil under conditions of low temperatures.

[Transformation of humic substances of highly oxidized brown coal by the basidiomycetes fungi Trametes hirsuta and Trametes maxima].

The ability of the white rot basidiomycetes Trametes hirsuta and Trametes maxima to transform coal humic substances (HS's) under the conditions of solid phase cultivation in the presence or absence of an easily available source of corbon (glucose) has been studied. It was shown that during the growth of the fungal strains used in media, containing HS's, destructive and condensation processes of HS transformation proceeded simultaneously. Based on a comparative physicochemical analysis of the initial HS's and HS's transformed by the fungi, it was established that, despite the introduction of glucose may favor a deeper transformation of HS's by basidiomycetes, the general direction of their modification is dominant reduction or oxidation and is determined by the physiological biochemical peculiarities of the strain used.

Comparative proteomic study of the basidiomycete Trametes hirsuta grown on different substrates.

Protein profiles of the basidiomycete Trametes hirsuta grown on standard medium without laccase as an inducer and on medium supplemented with CuSO4 were analyzed using a differential proteomics approach. Protocols developed for isolation and purification of extracellular and intracellular proteins of the mycelium allowed us to show extensive extraction of protein components. Simultaneously, components hampering two-dimensional electrophoresis (pigments, low molecular mass metabolites) were removed from the samples, and high-resolution protein maps were obtained. Analysis of the basidiomycete secretomes revealed qualitative changes in the protein profile: the addition of CuSO4 as an inducer resulted in increase in the produced laccase isoforms and/or isozymes from 7 to 11, whereas its pI range change exceeded 2 units. The number of separated intracellular protein components was 552 and 502 for the control medium and medium with the inducer, respectively. Comparative analysis of the protein maps revealed five regions with the most pronounced differences in the protein profiles. The proteins of interest were identified using MALDI-TOF/TOF mass spectrometry with subsequent peptide fingerprinting. Some intracellular proteins (ß-subunits of ATP synthase, molecular chaperones, chaperone activator) upregulated during the growth on the inducer-containing medium were identified. These proteins are supposed to be involved in the regulation of laccase biosynthesis during folding and secretion of the enzyme.

[Comparative analysis of the ligninolytic potential of basidiomycetes belonging to different taxonomic and ecological groups].

Screening of the ligninolytic activity of basidiomycetes from the Culture Collection of the Komarov Botanical Institute, Russian Academy of Sciences, belonging to diterent taxonomic and ecological groups was performed. The patterns of the position of taxa of active producers of ligninolytic enzymes in the modern system of fungi were identified. Cluster analysis showed that the group of fungi with the greatest lign- inolytic and degradation potential includes representatives of the families Pleurotaceae, Polyporaceae, and Phanerochaetaceae, which perform the first stages of wood decomposition. As a result, species of interest for the further study of their oxidative potential and use in biotechnology were selected.

[Range of Gene candidates involved in biosynthesis of laccase from basidiomycete Trametes hirsuta].

A comparative analysis of transcripts from the basidiomycota T. hirsuta grown with and without an inducer of the laccase biosynthesis was carried out. Methods of subtraction hybridization and massive parallel sequencing were used for this purpose. Unique transcripts encoded by genes that have a relatively high level of expression and belong to different gene ontology categories were identified. Also, a large number of transcripts were found to encode for predicted proteins, as well as noncoding transcripts. The latter may represent regulatory RNA molecules. Transcripts that increase their abundance when the laccase synthesis is induced are selected as gene-candidates involved in the laccase biosynthetic pathway.

Purification, biochemical characterization, and structure of recombinant endo-1,4-ß-xylanase XylE.

The gene xylE encoding endo-1,4-ß-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding ß-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ(1/2) of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K(m)) and 75 µmol/min per mg (V(max)) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 Å resolution. The 3D structure of an endo-1,4-ß-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.

[Analysis of functional properties of biologically active substances using eukaryotic cell models (review)].

This work presents an analysis of current data on the investigation into the functional properties of biologically active substances in model systems based on cultivated human cells. The knowledge regarding the practical application of cell cultures for the analysis of functional properties of bioactive substances is summarized, including antioxidant, immunomodulating, pro- and prebiotics, and chemoprevention properties. The most promising directions in cell culture model development for the investigation of functional properties, including three-dimensional models, are discussed.

[Use of basidiomycetes in industrial waste processing and utilization technologies: fundamental and applied aspects (review)].

This review provides an analysis of recent data on the mechanisms of degradation of lignocellulosic materials and xenobiotics by basidiomycetes. Special attention is given to the analysis of the current state of research of ligninolytic enzymes and their involvement in the degradation ofxenobiotics. Data on the practical use of basidiomycetes for bioconversion of industrial wastes are systematized. The most promising areas of bioconversion technologies are considered, such as contaminated water purification (including wastewater), cleanup of soils contaminated with heavy metals and xenobiotics, and degradation of difficult-to-degrade substrates (lignin and lignocellulose wastes, low-energy coal, and synthetic polymers).

[A heterologous production of the Trametes hirsuta laccase in the fungus Penicillium canescens].

A heterologous protein expression in the fungus Penicillium canescens is described for the first time. The fungal strains producing Trametes hirsuta laccase under control of a highly efficient promoter of the P. canescens gene bgaS has been constructed. These strains efficiently transcribe the T. hirsuta 072 laccase gene with a correct intron splicing. Activity of the secreted heterologous laccase in the culture liquid reaches 3 U/ml, accounting for 98% of the total laccase activity, which demonstrates a high efficiency ofheterologous secretion. The synthesized P. canescens laccase has the same molecular weight as the enzyme produced by T. hirsuta 072.

[Extremophilic microorganisms: biochemical adaptation and biotechnological application (review)].

The analysis of modern data on biochemical adaptation of microorganisms for living in extreme conditions is presented in this review. Special attention has been paid to the analysis of adaptive responses of microorganisms to the conditions of increased radiation at the molecular and cellular levels. The data on the practical application of extremophils and extremoenzymes, synthesized by them, biologically active substances, biopolymers and so on, have been systematized.

Enzymological properties of endo-(1-4)-beta-glucanase Eg12p of Penicillium canescens and characteristics of structural gene egl2.

Gene egl2 of secreted endo-(1-4)-beta-glucanase of glycosyl hydrolase family 5 of the mycelial fungus Penicillium canescens was cloned. The gene was expressed in P. canescens under control of a strong promoter of the bgaS gene encoding beta-galactosidase of P. canescens, and endoglucanase producing strains were obtained. Chromatographically purified recombinant 48 kDa protein had pH and temperature optima 3.4 and 60 degrees C, respectively, exhibited specific activity of 33 IU, and had K(m) and V(max) in CM-cellulose hydrolysis of 10.28 g/liter and 0.26 micromol/sec per mg, respectively.

Effect of solvent phase transitions on enzymatic activity and structure of laccase from Coriolus hirsutus.

The effect of solvent phase transitions on catalytic activity and structure of the active site of laccase produced by the Basidiomycetes Coriolus hirsutus 072 was studied. As shown by small-angle X-ray scattering, laccase exists in solution as a mixture of monomeric and aggregated particles in the percent ratio 85:15. This ratio did not change on phase transitions. A complex nature of laccase activity dynamics during thawing and further heating to 20 degrees C was shown. Spontaneous oxidation of T1 copper center in the temperature range 12-20 degrees C was not observed. According to spectral data, the structure of laccase active sites including all copper centers of types T1, T2, and T3 changes during the phase transition.

[Cloning of the Penicillium canescens endo-1,4-beta-glucanase gene egl3 and the characterization of the recombinant enzyme].

The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-beta-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for beta-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54 degrees C, respectively. The Km and Vm values for CMC hydrolysis were determined; they amounted to 17.1 g/1 and 0.31 microM/(mg s), respectively.

[Enzymatic hydrolysis of keratin-containing stock for obtaining protein hydrolysates].

Optimization of the process of enzymatic hydrolysis of keratin-containing stock aimed at obtaining hydrolysates of high biological value has been performed. The increasing of the stock/water weight ratio, the amount of the alkaline protease preparation from Acremonium chrysogenium added and the temperature of the reaction mixture resulted in an increase in the yield and antioxidant capacity of hydrolysis products. The molecular masses of soluble products obtained under optimal hydrolysis conditions ranged from 3.55 to 3.60 kDa. High antioxidant capacity, 100% bioavailability and a well-balanced amino acid composition was characteristic of the hydrolysis products.