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.

[The choice of antibiotic in microbiological analysis of phlegm.]

The efficiency of application of the test-system "Vy`borAntibiotika" (AntibioticChoice) in incubation of a maximal possible number of bacteria from pathologic material in case of pneumonia was studied. The results of meta-genome analysis permitted to establish that test-system support incubation of practically all bacteria detected in phlegm, including those attributed to so far non-incubated ones. The comparison of the results was carried out concerning a standard detection of sensitivity of bacteria to antibiotics and choice of efficient medicinal according the results of application of test-system "Vy`borAntibiotika". The obtained data demonstrates that test-system permits to choose antibiotic during 6-20 hours without isolation of pure strain.

[The choice of antibiotic in microbiological study of pyoinflammatory processes.]

The efficiency of application of test-system "Antibiotic Choice" was examined concerning evaluation of sensitivity to medications of maximal possible number of bacteria from pathological samples at burn trauma without isolation of pure culture. The results of metagenome analysis demonstrated that test-system permits supporting factually all bacteria discovered in wound discharge, including ones related to not cultivated yet. The comparison was carried out concerning results of standard identification of sensitivity of bacteria to antibiotics and efficient medication according the results of application of test-system "Antibiotic Choice". The obtained results demonstrate that test-system permits choosing antibiotic during 6-20 hours without separation of pure culture.

[CHOOSING ANTIBIOTIC IN MICROBIOLOGICAL ANALYSIS OF INFECTIONS OF URINARY EXCRETION SYSTEM].

The effectiveness of application of test-system "Choice of antibiotic" was evaluated as a tool for incubation of maximal amount of bacteria from pathological material under acute cystitis. The results of meta-genome analysis established that test-system permits supporting growth of practically all bacteria detected in urine, including ones relating to "uncultivated for the present". The comparison of results of standard detection of sensitivity of bacteria to antibiotics and identification of effective pharmaceutical according the results of application of test-system "Choice of antibiotic" as well was implemented It is demonstrated that test- system permits choosing antibiotic during 6-20 hours wiihout isolation of pure strain.

Time course analysis of mechanical ventilation-induced diaphragm contractile muscle dysfunction in the rat.

Controlled mechanical ventilation (CMV) plays a key role in triggering the impaired diaphragm muscle function and the concomitant delayed weaning from the respirator in critically ill intensive care unit (ICU) patients. To date, experimental and clinical studies have primarily focused on early effects on the diaphragm by CMV, or at specific time points. To improve our understanding of the mechanisms underlying the impaired diaphragm muscle function in response to mechanical ventilation, we have performed time-resolved analyses between 6 h and 14 days using an experimental rat ICU model allowing detailed studies of the diaphragm in response to long-term CMV. A rapid and early decline in maximum muscle fibre force and preceding muscle fibre atrophy was observed in the diaphragm in response to CMV, resulting in an 85% reduction in residual diaphragm fibre function after 9-14 days of CMV. A modest loss of contractile proteins was observed and linked to an early activation of the ubiquitin proteasome pathway, myosin:actin ratios were not affected and the transcriptional regulation of myosin isoforms did not show any dramatic changes during the observation period. Furthermore, small angle X-ray diffraction analyses demonstrate that myosin can bind to actin in an ATP-dependent manner even after 9-14 days of exposure to CMV. Thus, quantitative changes in muscle fibre size and contractile proteins are not the dominating factors underlying the dramatic decline in diaphragm muscle function in response to CMV, in contrast to earlier observations in limb muscles. The observed early loss of subsarcolemmal neuronal nitric oxide synthase activity, onset of oxidative stress, intracellular lipid accumulation and post-translational protein modifications strongly argue for significant qualitative changes in contractile proteins causing the severely impaired residual function in diaphragm fibres after long-term mechanical ventilation. For the first time, the present study demonstrates novel changes in the diaphragm structure/function and underlying mechanisms at the gene, protein and cellular levels in response to CMV at a high temporal resolution ranging from 6 h to 14 days.

Mass spectrometric study of bradykinin-related peptides (BRPs) from the skin secretion of Russian ranid frogs.

Amphibian skin secretion is known to contain biologically active peptides. Bradykinins and related peptides (BRPs) can be found in these animals, while frogs from the genus Rana are considered to be leaders in the levels and variety of these peptides. A reasonable rationalization of this fact is that bradykinins are efficient defense compounds against predators. Forty-four various BRPs have been identified in the skin secretions of five ranid frog species (R. ridibunda, R. lessonae, R. esculenta, R. temporaria, R. arvalis) from the Zvenigorod region (Moscow district, Russia). Some of these peptides are already known, but the novel ones constitute a significant portion. An interesting group of novel peptides was isolated from R. lessonae. These are bradykinin analogues bearing a tyrosine residue in the 5th or 8th position. [Arg(0), Trp(5), Leu(8)]bradykinin and [Thr(6), Leu(8)]bradykinin that had been isolated from fish and avian species, respectively, were also detected in the frog secretion, supporting the predator defense hypothesis. Furthermore, a novel group of BRPs named 'lessonakinins' was discovered in R. lessonae and R. esculenta. All of them include the [Arg(0), Trp(5), Leu(8)]bradykinin sequence and have some structural resemblance to the precursor of this peptide cloned by Chen and coworkers recently. However, the C-terminal part of the lessonakinins does not match the sequence predicted by Chen, demonstrating possible incompleteness of information obtained by cDNA cloning.

Novel natural peptides from Hyla arborea schelkownikowi skin secretion.

Hyla arborea schelkownikowi is one of the leaf frog species inhabiting the southern territories of Russia and the former USSR. This frog species is a member of the Hylidae Rafinesque, 1815 batrachians family. The present study deals with the previously uninvestigated peptidome of the Hyla arborea schelkownikowi skin secretion. Nano-electrospray ionization Fourier transform mass spectrometry (nanoESI-FTMS) of the skin secretion, in the intact form and after acetylation, was selected as the general method of analysis. Electron-capture dissociation (ECD) and collision-induced dissociation (CID) fragmentation were both employed, while de novo sequencing was performed by manual interpretation of the MS data. The suppression of the cyclization of b-ions in the mass spectrometer by the acetylation reaction proved to be very efficient for the de novo sequencing of short peptides. Ten skin peptides were found and all of them, except for bradykinin, had not previously been reported. Six of the peptides belong to the tryptophyllins and related peptides, while three peptides are similar to the aureins.

Binding of sterols affects membrane functioning and sphingolipid composition in wheat roots.

The present work was devoted to the exploration of the role of sterols in the functioning of membranes in root cells. Membrane characteristics and composition of the membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with exogenous cholesterol and antibiotic nystatin, which specifically binds with endogenous sterols, were analyzed. Cholesterol caused a fall of membrane potential, acidification of the incubation medium, decrease in potassium leakage of roots, and increase in the level of exogenous superoxide radical. Similarly to cholesterol, the application of nystatin also induced the depolarization of the plasma membrane, but in contrast with cholesterol it was accompanied by alkalinization of the incubation medium and decrease in the level of exogenous superoxide radical. Analysis of membrane lipids showed that following nystatin treatment the total sterol content in roots did not change, while the level of complex sphingolipids represented mainly by glycoceramides became higher. Using mass spectrometry with electrospray ionization ((+)ESI-MS) for the analysis of the glycoceramide composition, we showed that nystatin induced changes in the ratios of molecular species of glycoceramides. It was suggested that the modification of the sterol component of plasma membrane could influence membrane functioning by changing the sphingolipid composition of lipid rafts.

[Effect of exogenic proteolytic enzymes on transfer of plasmid genes in mixed bacterial biofilms].

The effect of the enzyme complex from Vobenzin on transfer of plasmid genes in biofilms of gramnegative bacteria was studied. The extracellular matrix of the bacterial biofilms was shown to contain extracellular DNA carrying the antibiotic resistance markers. The action of the enzyme complex resulted in lower frequency of the antibiotic resistance genes transfer in mixed bacterial biofilms.

Electrospray ionization tandem mass spectrometry sequencing of novel skin peptides from Ranid frogs containing disulfide bridges.

Tandem mass spectrometry sequencing, as well as Edman sequencing of peptides belonging to the Rana genus, represents a difficult task due to the presence of a disulfide bridge at the C-terminus and their rather high molecular masses (over 2000 Da). The present study throws light upon the sequence of three rather long peptides (more than 20 amino acid residues each) isolated from the skin secretion of Russian frogs, Rana ridibunda and Rana arvalis. This novel aspect involves the fact that the sequences (including two sequences established de novo) were determined exclusively by means of mass spectrometry. A combination of electron capture dissociation (ECD) and collision-induced dissociaiton (CID) data accompanied by exact mass measurements (LTQ Fourier transform ion cyclotron resonance mass spectrometer) facilitated reaching the goal. To overcome the difficulty dealing with disulphide bridges ("Rana box"), reduction of the S-S bond with dithiotreitol followed by derivatization of Cys residues with iodoacetamide was used. The sequence was determined using combined spectral data on y and b series of fragment ions. A multiple mass spectrometry (MS(3)) experiment was also used to elucidate the sequence inside the "Rana box" after cysteine derivatization. Exact mass measurements were used to differentiate between Lys and Gln residues, while characteristic losses of 29 and 43 Da (d and w fragment ions) in CID and ECD experiments allowed us to distinguish between Ile and Leu isomeric acids.

[Combined action of antibiotics and deoxyribonuclease on bacteria].

The impact of DNase on formation of biofilms by grampositive and gramnegative bacteria was studied. The enzyme was shown to inhibit the biofilm formation and have no toxicity to bacteria. When applied to the formed associations, DNase potentiated the effect of antibiotics on the bacteria located in them. An increase in the antimicrobial effect of various nonrelated antibiotics was not connected with a change in the bacteria susceptibility but likely resulted from a break of the genetic cooperation of the cells' community, connected with functions of extracellular DNA of the biofilm's matrix.

[Enzyme DNA-ase use in patients with maxillofacial abscesses and phlegmons].

The results of the clinical studies indicate that DNAase treatment of the patients with maxillofacial abscesses and phlegmons increases the effect of antibacterial therapy. During microbiologic testing in the patients treated with DNAase together with antibacterial therapy it was recorded a marked decrease in the plating of bacteria. This effect is supposed to be connected with enzyme ability to penetrate into microbial biofilms and disturb microbial interactions.

Mass-spectrometry-based characterization of oxidations in proteins.

Protein modifications such as oxidations have a strong impact on protein function and activity in various organisms. High-resolution mass spectrometric techniques in combination with various sample preparation methodologies allow for the in-detail characterization of protein structures and strongly contribute to a greater understanding of the impact of protein modifications in nature. This paper outlines the general workflows for the characterization of oxidation sites in proteins by mass spectrometry (MS). Different types of oxidations are taken into consideration; both qualitative and quantitative aspects of MS-based approaches are presented with respect to oxidized proteins. Both bottom-up and top-down MS approaches are described and evaluated; a wide range of the particular applications corresponding to these techniques is also presented. Furthermore, the common advantages and downsides of these techniques are assessed. The approaches for enrichment of low-abundance oxidized proteins are extensively presented for different cysteine oxidations and protein carbonylations. A short description about databases and bioinformatic software solutions for oxidative protein prediction, identification, and biological interpretation is also given in this review.

[Impact of exogenic proteolytic enzymes on bacteria]. / Vliianie ékzogennikh proleoticheskikh fermentov na bakterii.

The impact of papain, trypsin and the enzyme complex vobenzyme on formation of biofilms by grampositive and gramnegative bacteria was studied. The enzymes were shown to inhibit the biofilm formation. When applilied to the formed associations, the enzymes potentiated the effect of antibiotics on the bacteria located in them. An increase in the antimicrobial effect of various nonrelated antibiotics was not connected with a change in the bacteria susceptibility but likely resulted from higher bioavailability of the drugs in the presence of the enzymes.

[Lymphotropic antibacterial therapy for preventing inflammatory complications of mandibular fractures]. / Limfotropnaia antibakterial'naia terapiia v profilaktike infektsionno-vospalitel'nykh oslozhnenii perelomov nizhnei cheliusti.

We propose a new method of preventing infectious and inflammatory complications of mandibular fractures crossing the dental arch. The method was based on regional lymphotropic administration of antibacterial drugs to the mastoid region of the temporal bone. This technique was applied in combined therapy of 51 patients. Comparative analysis with the control group revealed high efficacy of the given method. Basing upon clinical experience we worked out indications to the regional lymphotropic antibacterial therapy as a part of complex prophylaxis of posttraumatic mandible osteomyelitis.

Mass spectrometric study of peptides secreted by the skin glands of the brown frog Rana arvalis from the Moscow region.

A high-performance liquid chromatography nano-electrospray ionization Fourier transform mass spectrometry (HPLC/nanoESI-FTMS) approach involving recording of collision-activated dissociation (CAD) and electron-capture dissociation (ECD) spectra of an intact sample and two its modifications after performic oxidation and reduction followed by carboxamidomethylation helps to establish peptide profiles in the crude secretion of frog species at mid-throughput level, including de novo sequencing. The proposed derivatization procedures allow increasing of the general sequence coverage in the backbone, providing complementary information and, what is more important, reveal the amino acid sequence in the cystine ring ('rana box'). Thus purely mass spectrometric efficient sequencing becomes possible for longer than usual proteolytic peptides. Seventeen peptides belonging to four known families were identified in the secretion of the European brown frog Rana arvalis inhabiting the Moscow region in Russia. Ranatuerins, considered previously a unique feature of the North American species, as well as a new melittin-related peptide, are worth special mention. The developed approach was previously successfully used for the identification of peptides in the skin secretion of the Caucasian green frog Rana ridibunda.