[Possibility of application of extracellular protease of micromycet Aspergillus ochraceus VKM F-4104D for determination of protein C content in human blood plasma]. / Vozmozhnost' primeneniia vnekletochnoi proteazy mikromitseta Aspergillus ochraceus VKM F-4104D dlia opredeleniia soderzhaniia proteina S v plazme krovi cheloveka.

It was shown that the activator activity of protein C, determined in normal plasma using Aspergillus ochraceus protease, is comparable with the activity of commercial protease analogue from the South American copperhead venom (ProtacÒ). It was found that protease of A. ochraceus can be used to determine protein C in plasma with its reduced content similar to ProtacÒ. Comparison of the activator protein C activity of A. ochraceus protease and the commercial analogue showed some excess of the activator activity of the fungal preparation, which may make it a promising substitute for the snake activator in diagnostical kits for determining the protein C content in clinical laboratories.

[Morphological and Physiological Properties of the Micromycete Arthrobotrys longa, a Producer of Longolytin, a Proteolytic Complex with a Thrombolytic Effect].

Production of proteinases with plasmin-like and plasminogen-activating activities by a micromycete Arthrobotrys longa 1 was studied. Polycyclic growth of the producer in submerged cultures was observed, with an endogenous rhythm of the periods of intense microconidia formation alternating with the phases of drastic increase in the content of producing mycelium. The highest plasminogen-like and plasminogen-activating activities (up to 1000 and 500 cond. U/mL, respectively) were found to correlate with the polycyclic growth of the producer, coinciding with the stage of microconidia germination. Enhanced secretion of proteinases with plasminogen-like and plasminogen-activating activity was found to be associated with increased specific growth rate of A. longa l.

[Screening of Producers of Proteinases with Fibrinolytic and Collagenolytic Activities among Micromycetes].

Screening for producers of proteinases with fibrinolytic (plasmin-like and plasminogen-activating) and collagenolytic activities-was carried out among 83 strains of microscopic fungi belonging to various ecological groups. Entomopathogenic micromycetes secreted proteinases with higher fibrinolytic and collagenolytic activity than saprotrophic, potentially phytopathogenic, and epiphytic strains. Micromycete strains possessing proteolytic enzymes with collagenase activity were revealed, as well as the strains producing proteinases with plasmin-like activity. None of the strains studied secreted proteinases possessing only plasminogen-activating activity. Tolypocladium inflatum k1 was found to be a producer of extracellular proteinases with high plasminogen-activating, plasmin-like, and collagenolytic activities. The specific plasminogen-activating activity of T. inflatum k1 was shown to be 20% higher than its plasmin-like activity.

[Properties of extracellular proteinase--an activator of protein C in blood plasma formed by Aspergillus ochraceus].

The properties of an extracellular proteinase activating plasma protein C isolated from the culture supernatant of A. ochraceus VKM F-4104D have been studied. This enzyme demonstrated a substrate specificity absent of hydrolyzing activity toward chromogenic proteinase substrates. On the basis of inhibitory analysis, the protein C-activating proteinase from A. ochraceus VKM F-4104D appeared to be a serine proteinase, together with that isolated from the venom of Agkistrodon contortrix contortrix. The isolated enzyme was a nonglycosylated protein with a molecular weight of about 33 kDa, pI 6.0 with an observed optimal activity under a pH of 8.0-9.0 and 37°C. A comparison of the properties of the protein C-activating proteinase formed by A. ochraceus and the enzyme derived from the venom of Agkistrodon contortrix contortrix demonstrated a similarity in their properties; however, proteinase from the micromycete appeared to be in the nonglycosylated state and possessed the ability to hydrolyze the chromogenic plasmin substrate H-D-Val-Leu-Lys-pNA.

[Identification of Target Extracellular Proteases--Activators of Proteins of Haemostasis System Produced by Micromycetes Aspergillus ochraceus and Aspergillus terreus].

Effects of extracellular proteases of Aspergillus ochraceus and Aspergillus terreus on plasma hemostasis proteins, consist of initiating the activation of prothrombin complex proteins, was detected. Was discovered, that A. ochraceus proteases have a direct influence on protein C and coagulation factor X, and A. terreus proteases causes their activation indirectly through kallikrein system stimulation. The ability of extracellular proteases of micromycetes activate prekallikrein in human blood plasma on the example of A. terreus was first demonstrated.

[Solid-State and membrane-surface liquid cultures of micromycetes: specific features of their development and enzyme production (a review)].

Specific features in the development of micromycetes, typical mechanisms of their enzyme production, and conditions providing for an increase in enzyme secretion by the microscopic fungi in solid-state (on natural substrates and inert carriers) and membrane-surface liquid cultures are considered. The prospects and advantages of these fermentation methods for the production of extracellular enzymes are discussed and compared with submerged cultures.

[Effect of extracellular proteases of micromycetes of the genus Aspergillus on proteins of haemostasis system].

Screening of the genus Aspergillus micromycetes for secretion of extracellular proteolytic enzymes, capable of acting on human proteins of the hemostatic system, has been conducted. The ability of extracellular proteases of Aspergillus to cleave specific proteins of the hemostatic system chromogenic peptide substrates, as well as activate a series of proenzymes (protein C, factor X and prothrombin) has been found. The ability of extracellular proteases of micromycetes activate X factor of human blood plasma was first shown at the first time.

[Production of extracellular proteinases (protein C activators in blood plasma) by the micromycete Aspergillus ochraceus during submerged and solid-state cultivation].

The conditions for the submerged and solid-state cultivation of the micromycete Aspergillus ochraceus VKM F-4104D, producing extracellular proteinases that activate protein C in human blood plasma, were optimized. It is shown that the protein C-activating activity of the micromycete in a solid-state culture was 1.5-3.5 times higher than in a submerged culture (as calculated per milliliter of culture medium). Among the extracellular proteins secreted by A. ochraceus VKM F-4104D during submerged and solid-state cultivation, a protein C-activating proteinase with a pI of 6.0-6.3 was identified.

[Aspergillus ochraceus myxomycetes produce extracellular proteinases--protein C activators of blood plasma].

Natural isolates of Aspergillus ochraceus myxomycetes from soil and plant remains from various regions have been screened. The isolated strains were characterized by similar cultural and morphological features and an identical nucleotide sequence in the ITS1-5,8S-ITS2 region of rDNA. The ability of the extracellular proteinases of A. ochraceus myxomycetes to activate protein C of blood plasma has been established. Differences are revealed in the accumulation of proteinases activating protein C and proteinases with thrombin- and plasmin-like activities in the growth dynamics of producers.

[Development and population structure of mixed (S + M) Pseudomonas aeruginosa cultures in the late stationary growth phase].

Population growth, the ratio between dissociants, pH, and levels of reducing sugars in the medium were monitored during prolonged (375 h) batch cultivation of Pseudomonas aeruginosa S and M dissociants on mineral medium with glucose. During the stationary growth phase (100-375 h), two scenarios were possible. The first one included extensive cell autolysis coupled to alkalinization of the medium and an increased ratio of the M dissociant. In the second case, acidification of the medium was coupled to the oscillating secondary growth, mostly of the M dissociant; the dynamics of cell numbers of this dissociant correlated with the dynamics of the culture optical density. In this scenario, periodical appearance of reducing sugars in the medium was detected; it was in the opposite phase with the changes of the M dissociant cell numbers. The differences between scenarios of P. aeruginosa growth in the late stationary phase were probably due to the physiological and biochemical characteristics of the S and M dissociants, including different pathways of glucose utilization (respiration or fermentation), resistance to acidification, synthetic (proteolytic) activity, and productivity of autoinducers.

[A comparison of the properties of bacteriocins formed by Lactococcus lactis subsp. lactis strains of diverse origin].

Bacteriocins formed by four strains of Lactococcus lactis subsp. lactis have been studied and compared: 729 (a natural strain isolated from milk), 1605 (a mutant of strain 729), F-116 (a recombinant obtained by fusing of protoplasts of the two related strain 729 and 1605), and a nisin-forming strain obtained by adaptive selection at Moscow State University. Antimicrobial activity studies revealed differences between the strains in the effects on individual groups of microorganisms; the activities of the strains were also distinct from that of Nisaplin (a commercial preparation of the bacteriocin nisin). Methods for isolation and purification of bacteriocins have been developed, making it possible to obtain individual components of antibiotic complexes as chromatographically pure preparations. Bacteriocins formed by the strains of Lactococcus lactis subsp. lactis have been identified and differences in their biological and physicochemical properties, established. A novel potent broad-spectrum antibiotic substance distinct from nisin has been isolated from the recombinant strain F-116.

[Variability of nisin-synthesizing wild strain Lactococcus lactis subsp. lactis 119 under the effect of ultraviolet light].

To increase the nisin synthesizing activity of the natural strain Lactococcus lactis subsp. lactis 119 isolated from sour milk, UV irradiation in different doses was used followed by isolation of productive clones. The highest mutation effect was observed with the dose of 76,000 erg/mm2, when 11% of the cells increased their productivity by 12.8% at the minimum survival rate. Two-step UV irradiation and adaptive selection on the nisin-contaning medium provided isolation of a strain with the activity 42.6% higher than that of the initial strain (3850 IU/ml). Natural and UV-induced variability of the strain by the nisin synthesis, growth rate, carbohydrate consumption and sensitivity to antibiotics of various groups were studied.

[Concentration on Diapak C 16 capsules of lovastatin, mevinolinic acid and other inhibitors of biosynthesis of sterins produced by Penicillium citrinum 89]. / Kontsentrirovanie lovastatina, mevinolinovoi kisloty i drugikh ingibitorov biosinteza sterinov, obrazuemykh Penicillum citrinum 89, na patronakh Diapak C 16.

The method of lovastatine and mevinolinic acid known as competitive inhibitors of HMG-CoA-reductase and produced by micromycetes was elaborated. The inhibitors from diluted water solutions were fully absorbed on Diapak C16 patrons. The rate of inhibitors elution from the patrones was more than 95 per cent. Patrons may be used for concentration of lovastatine group inhibitors from the culture media. Inhibitors synthesis by the Penicillium citrinum 89 was investigated in dynamics with the use of Diapak C16 patrones. It was shown that UV-spectrum of inhibitor produced by P. citrinum 89 was identical with compactin spectrum and had absorbance maximum at 230, 237 and 247 nm.

[Micromycetes metabolites--inhibitors of growth and sterol biosynthesis in yeasts]. / Metabolity mikromitsetov--ingibitory rosta i biosinteza sterinov u drozhzhei.

Antifungal activity of micelial fungus metabolites (of genera Aspergillus, Penicillium, Fusarium, Stachybotris, Cladosporium, Alternaria, Gliocladium, Paecilomyces, Trichoderma etc.) was determined. It was shown that antifungal activity of some micromycetes is due to the formation of substances inhibiting sterols biosynthesis in eucaryote cells. Inhibitors of enzymes of sterols biosynthesis were isolated and their activity was investigated. It was shown, that isolated fungus inhibitors of sterols biosynthesis inhibited the growth of test-organism Rhodotorula rubra and decreased ergosterin level in yeast cells. The qualitative content of yeast cell sterols was not changed in the presence of fungus inhibitors.

[Preparations of extracellular proteinases from Aspergillus ochraceus 513 and Aspergillus alliaceus 7dN1]. / Izuchenie preparatov vnekletochnykh proteinaz gribov Aspergillus ochraceus 513 i Aspergillus alliaceus 7 dN1.

Preparations of extracellular proteolytic enzymes with high anticoagulant activity resembling protein C activators were isolated from the culture liquids of Aspergillus ochraceus 513 and Aspergillus alliaceus 7 dN1 by precipitation with ammonium sulfate and subsequent purification from ammonium ions by gel filtration on a column with Sephadex G-25. The pH and temperature activity optima and stability of the proteolytic enzymes from A. ochraceus 513 and A. alliaceus 7 dN1 were determined.

Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus.

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.

[Isolation, purification, and separation of the complex preparation of extracellular proteinases with fibrinolytic and anticoagulant properties from Aspergillus ochraceus 513]. / Vydelenie, ochistka i razdelenie kompleksnogo preparata vnekletochnykh proteinaz Aspergillus ochraceus 513 s fibrinoliticheskimi i antikoaguliantnymi svoistvami.

The extracellular proteinase complex of the microscopic fungus Aspergillus ochraceus 513 was isolated, purified, and separated by affinity chromatography on bacillichin-silochrom and subsequent column chromatography on DEAE-Toyopearl 650 M. The extracellular enzyme of the protein C activator type had a molecular mass of 36.5 kDa and activity close to that of the Agkistrodon snake venom protein C activator. The fibrinolytic and anticoagulant activities of the enzyme were investigated.

[Inhibition of bacterial bioluminescence by chlorophenols]. / Ingibirovanie bakterial'noi bioliuminestsentsii khlorfenolami.

Photobacteria were used as a test object for rapid monitoring of ecotoxicants. Specific inhibitory effects of phenol and its chlorinated derivatives (2-chlorophenol, 2,3-dichlorophenol, pentachlorophenol, 2,4-dichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxyacetic acid) on bioluminescence and respiration of intact cells, as well as on the emission activity of the bioluminescence system and luciferase itself, were studied. The toxic effect on the photobacterial cells was found to increase as the number of chlorine atoms in the chlorophenol molecule increases. However, this trend was not observed in cell-free systems (purified luciferase or the protein fraction of a cell-free extract treated with (NH)4SO4 at 40-75% saturation). Bacterial cells have a higher threshold sensitivity to chlorophenols in comparison to the sensitivity of the bioluminescence enzyme system or luciferase. Neutral phenols inhibit luciferase by competing with decanal, whereas a mixed mechanism of inhibition with this substrate is typical of phenoxyacetic acids. With respect to FMNH2, all chlorophenols tested in this work were uncompetitive inhibitors. Oxygen uptake by photobacteria was shown to be insensitive to chlorophenols, at least within the concentration range that was effective in bioluminescence inhibition. The results of this study suggest that bacterial bioluminescence system is not the primary target of the chlorophenol-induced effect on photobacteria.