Expression of coagulin A with low cytotoxic activity by Pediococcus pentosaceus ST65ACC isolated from raw milk cheese.

AIMS: We aimed to evaluate some specific conditions for growth of Pediococcus pentosaceus ST65ACC and its bacteriocin expression through ABC transporters; to purify the bacteriocin and determine its sequence; and to evaluate the cytotoxicity potential of the purified bacteriocin(s). METHODS AND RESULTS: The results presented for growth behaviour of P. pentosaceus ST65ACC showed that the bacterial growth was slightly influenced when cultured in MRS broth with different amounts of inoculum: 1, 2, 5 and 10%. The bacteriocin activity increased when 5 and 10% inocula were used. The carbon source (glucose) used in different amounts (1, 2, 3 or 4%) had no significant effect on growth and bacteriocin production. The studied strain P. pentosaceus ST65ACC was able to metabolize xylooligosaccharide (XOS) as the sole carbon source, resulting in the production of an antimicrobial peptide. The genes involved in the ABC transport system and sugar metabolism of P. pentosaceus ST65ACC were expressed at different levels. The bacteriocin produced by P. pentosaceus ST65ACC was partially purified by precipitation with ammonium sulphate (40% saturation), followed by reversed-phase liquid chromatography, resulting in the identification of an active bacteriocin. Tandem mass spectrometry was used to identify the partial sequence KYYGNGVTCGKHSCSVDWGK sharing high similarity to coagulin A. The semi-purified bacteriocin had low cytotoxicity based on estimated values for maximal nontoxic concentration (MNC) and cytotoxicity concentration (CC50 ). CONCLUSIONS: The bacteriocin produced by P. pentosaceus ST65ACC is similar to coagulin, with low cytotoxicity, strong antimicrobial activity and possible additional metabolite routes in the producer cell. In addition to MRS broth, bacteriocin was produced also in medium containing XOS (as the single carbon source). SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of evaluation of the role of ABC transporters in the expression of bacteriocin by P. pentosaceus, cultured in MRS and XOS.

Purification and characterization of the bacteriocin produced by Lactobacillus sakei MBSa1 isolated from Brazilian salami.

AIMS: The study aimed at determining the biochemical characteristics of the bacteriocin produced by Lactobacillus sakei MBSa1, isolated from salami, correlating the results with the genetic features of the producer strain. METHODS AND RESULTS: Identification of strain MBSa1 was performed by 16S rDNA sequencing. The bacteriocin was tested for spectrum of activity, heat and pH stability, mechanism of action, molecular mass and amino acid sequence when purified by cation-exchange and reversed-phase HPLC. Genomic DNA was tested for bacteriocin genes commonly present in Lact. sakei. Bacteriocin MBSa1 was heat-stable, unaffected by pH 2·0 to 6·0 and active against all tested Listeria monocytogenes strains. Maximal production of bacteriocin MBSa1 (1600 AU ml(-1)) in MRS broth occurred after 20 h at 25°C. The molecular mass of produced bacteriocin was 4303·3 Da, and the molecule contained the SIIGGMISGWAASGLAG sequence, also present in sakacin A. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins T-α, T-ß, X, P, G and Q). CONCLUSIONS: In the studied conditions, Lact. sakei MBSa1 produced sakacin A, a class II bacteriocin, with anti-Listeria activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study covers the purification and characterization of the bacteriocin produced by a lactic acid bacteria isolated from salami (Lact. sakei MBSa1), linking genetic and expression information. Its heat-resistance, pH stability in acid conditions (pH 2·0-6·0) and activity against L. monocytogenes food isolates bring up a potential technological application to improve food safety.

Genomic, metabolomic, and functional characterisation of beneficial properties of Pediococcus pentosaceus ST58, isolated from human oral cavity.

Bacteriocins produced by lactic acid bacteria are proteinaceous antibacterial metabolites that normally exhibit bactericidal or bacteriostatic activity against genetically closely related bacteria. In this work, the bacteriocinogenic potential of Pediococcus pentosaceus strain ST58, isolated from oral cavity of a healthy volunteer was evaluated. To better understand the biological role of this strain, its technological and safety traits were deeply investigated through a combined approach considering physiological, metabolomic and genomic properties. Three out of 14 colonies generating inhibition zones were confirmed to be bacteriocin producers and, according to repPCR and RAPD-PCR, differentiation assays, and 16S rRNA sequencing it was confirmed to be replicates of the same strain, identified as P. pentosaceus, named ST58. Based on multiple isolation of the same strain (P. pentosaceus ST58) over the 26 weeks in screening process for the potential bacteriocinogenic strains from the oral cavity of the same volunteer, strain ST58 can be considered a persistent component of oral cavity microbiota. Genomic analysis of P. pentosaceus ST58 revealed the presence of operons encoding for bacteriocins pediocin PA-1 and penocin A. The produced bacteriocin(s) inhibited the growth of Listeria monocytogenes, Enterococcus spp. and some Lactobacillus spp. used to determine the activity spectrum. The highest levels of production (6400 AU/ml) were recorded against L. monocytogenes strains after 24 h of incubation and the antimicrobial activity was inhibited after treatment of the cell-free supernatants with proteolytic enzymes. Noteworthy, P. pentosaceus ST58 also presented antifungal activity and key metabolites potentially involved in these properties were identified. Overall, this strain can be of great biotechnological interest towards the development of effective bio-preservation cultures as well as potential health promoting microbes.

[Functional properties of endothelium of peripheral vessels in adolescents].

In a random sample of adolescent schoolchildren (n=204) we assessed functional state of endothelium of brachial artery by test with reactive hyperemia. The data obtained was compared with values of arterial pressure and nutritional status of these adolescents and results of questionnaire survey of their parents concerning heredity and lifestyle. In 12.4% of examined subject values of flow mediated vasodilation were abnormal (less than 10%). We found that these abnormalities were most often observed in adolescents with hereditary predisposition to cardiovascular diseases, elevated arterial pressure, excess of fatty mass, inactive way of life.

[About the evaluation of school children's health].

The information from primary medical documentation and questionnaires of children and their parents concerning the health of children of 12-14 years old is compared. The lack of correspondence is marked between the subjective perception of health (both children and their parents) and the results of comprehensive evaluation of health status based on the medical criteria. The more detailed analysis permitted to mark out a number of possible causes of such a lack of correspondence: absence of changes in general state among children with compensated chronic pathology, subjective overstating of estimate of real level of health by children and parents, hypo diagnostics of certain pathologic states in school children by medical professionals. The results revealed the need in more close attention of pediatricians to the child subjective self-perception of health, active identification of complaints and pathologic symptoms during dispanserization examinations of schoolchildren.

[The medical provision of schoolchildren in the children educational institutions].

The major issues of the medical follow-up in the children educational institutions are discussed. The data related to the need of the development and enhancement of preventive course is presented. The main possible ways to resolve the existing problems are enumerated. The necessity of implementing the new screening diagnostical technologies during the procedure of preventive examinations is proved. The using of the efficient diet and the physical education, the development of the dominant of healthy life style and the targeted preventive activities among children of medical social, medical psychological and biological risk groups is substantiated.

[The course and development of epilepsy in patients with typical variant of Rett syndrome and mutations]. / Osobennosti épilepsii u detei s tipichnym variantom sindroma Retta, vyzvannym mutatsiei.

AIM: To study the anamnesis, clinical state, electro-encephalographic and brain MRI characteristics in patients with Rett syndrome (МЕСР2) and epilepsy. MATERIAL AND METHODS: Eleven female patients, aged from 3 to 23 years, with Rett syndrome and MeCP2 mutations were studied. The study continued for 10 years (2006-2015). Assessment of neurological and mental status, night sleep video-EEG monitoring, MRI were performed. RESULTS AND CONCLUSION: Epilepsy was diagnosed in six cases (54.5%). Mean age at onset of epileptic seizures was 3 years 9 month. The following types of seizures were described: generalized, myoclonic, myotonic, tonic, versive, focal motor, atypical absences. Status epilepticus developed in one patient. Generalized seizures were identified in 56.25%, focal seizures in 43.75%. EEG changes were found in 9 patients (81.8%): slowing of the activity, episodes of periodic regional slowing, regional epileptiform activity and diffuse epileptiform activity, benign focal epileptiform discharges (BFED) of childhood, multiregional epileptiform activity. Five patients were treated with antiepileptic drugs. All of them had improved during treatment: a reduction of frequency of seizures was up to 50% in 4 cases (80%). One patient with resistant epilepsy was treated with the combination of drugs (levetiracetam, topiramate, zonisamide, benzodiazepine) that led to stopping of seizures during night sleep and decrease in the frequency of daytime seizures by 50%. Further research of epilepsy and efficacy of antiepileptic drugs in Rett syndrome is required.

[The course and the development of epilepsy in patients with typical variant of Rett syndrome and mutations]. / Osobennosti techeniia i razvitiia épilepsii u detei s tipichnym variantom sindroma Retta, vyzvannogo mutatsiei.

AIM: Studying data of anamnesis, clinical state, electro-encephalographic, brain MRI in patients with Rett syndrome (МЕСР2). MATERIAL AND METHODS: We studied 11 patients (female) from three to 23 years old with Rett syndrome and MeCP2 mutations. Observation continued 10 years (2006-2015). We analyzed the results of the neurological status, night sleep video-EEG monitoring, MRI. RESULTS AND CONCLUSION: Epilepsy diagnosed in six cases (54, 5%). The overage age of debut of epileptic seizures was 3 years 9 months. There are some types of seizures: generalized, myoclonic, myotonic, tonic, versive, focal motor, atypical absences. Status epilepticus evolved in one patient. Generalized seizures were 56, 25%, focal seizures - 43, 75%. EEG changing marked in nine patients (81, 8%): slowdown back activity, episodes of periodic regional slowdown, regional epileptiform activity, and diffuse epileptiform activity like benign focal epileptiform discharges (BFED). five patients took antiepileptic drugs. All of them had improved during treatment. There were reducing of frequency of the seizures up 50% - 4 cases (80%). one patients with resistant epilepsy was taken combination of drugs (levetirecetam, topiromat, zonisamide, benzodiazepine) with stopping of seizures in the night sleep and decreasing of frequency of daytime seizures to 50%. We believe there is very important of study epilepsy in patients with Rett syndrome and improvement of its treatment.

[Clinical features and current laboratory diagnosis of leptospirosis of grippotyphosa serogroup].

AIM: To characterize persistence of the causative agent in the blood of patients with leptospirosis, to evaluate significance of the results of polymerase chain reaction (PCR) with isolation of ribosomal RNA (rRNA) and microagglutination reaction (MAR) in diagnosis of leptospirosis at different stages of the disease. MATERIAL AND METHODS: The study included 94 patients with leptospirosis: 51 (54.2%) males, 43 (45.8%) females, 74 (78. 7%) urban and 20 (21.3%) rural citizens in acute leptospirosis, early and late convalescence. Leptospirosis was diagnosed on the basis of clinico-epidemiological, laboratory-device data. All the patients were divided into 2 groups: with leptospiral jaundice (n = 33, 35.1%) and without leptospiral jaundice (n = 61, 64.9%). The disease of moderate severity was in 64 (68%) patients. Verification of the diagnosis was based on determination of specific antibodies titer in MAR using standard set of diagnostic cultures. PCR determined rRNA of leptospira in the blood on the disease day 5, 10 and 15, 1, 2, 3, 6 and 12 months after the disease onset. The results were processed with methods of parametric and non-parametric statistics. CONCLUSION: PCR demonstrated diagnostic value in early leptospirosis (sensitivity of the method in the disease week 1 was 86%). Diagnostic significance of MAR reached 81.8% later - in 3 weeks. Detection of leptospiral rRNA by PCR in the autopsy material allows one to recommend this method for additional verification of the diagnosis including as an alternative to bacterioscopic test.

Saline containing phosphatidylcholine liposomes possess the ability to restore endothelial function damaged resulting from gamma-irradiation.

The protective action of passive saline filled ("empty") phosphatidylcholine liposomes (PCL) on endothelial function was examined in thoracic aortas obtained from gamma irradiated (6 Gy) Chinchilla rabbits, and then verified in experiments on non-anesthetized and anesthetized rats. Acetylcholine (ACh)-induced vascular relaxant responses in isolated vascular tissues rats were used as the test of endothelial integrity and its functional ability. It was shown that when added to the bath solution (100 microg/ml), PCL effectively restored endothelium-dependent ACh relaxations of isolated vascular rings damaged resulting from gamma-irradiation but had no effect on endothelium-independent vascular responses to therapeutic nitric oxide (NO) donors. The liposomes were also without protective effect when injected to the rabbits intraperitoneally (30 mg/kg) 1 hour before irradiation. In contrast, PCL, being injected at the same dose 1 hour after radiation impact, promote normalization of both endothelium-dependent vascular responses to ACh and nitric oxide (NO) donors. PCL restored also the sensitivity of vascular tissues to authentic NO (aqueous NO solution) that was surprisingly increased after irradiation, and normalized relationship between ACh-stimulated NO release and relaxant response amplitudes in irradiated aortas. Experiments on non-anesthetized and anesthetized rats demonstrated that irradiation led to significant elevation in the level of arterial blood pressure without any changes in cardiac contractility. PCL administration (25 mg/kg, i.v.) effectively normalized an increased arterial blood pressure in irradiated animals. In conclusion, it appears that PCL due to its ability to normalize NO-dependent vascular tone control mechanisms might be worthwhile therapeutic approach in case of ionizing irradiation accident. These result support the concept that the depression of endothelium-dependent vascular responses after irradiation may be result of decreased NO bioavailability due to its conversion to less potent vasodilators during irradiation-induced oxidative attack.

Resistance to the polyether ionophore antibiotic pandavir (nigericin) in two producing strains of Streptomyces.

In two pandavir (nigericin) producing strains, Streptomyces hygroscopicus 155 and Streptomyces albogriseolus 444, an enzyme activity was detected leading to inactivation of the antibiotic in the presence of ATP-Na2. Apparently, the observed inactivation is specific for the antibiotic produced by these strains. The nigericin producing strains were also found to be less permeable to pandavir than their non-producing variants.

[The adaptive response of B cells to the action of cytotoxic preparations in culture. III. The possible mechanism of the action of adriamycin on immunoglobin synthesis by the cells of murine myeloma]. / Adaptivnyi otvet B-kletok na deistvie tsitotoksicheskikh preparatov v kul'ture III. Vozmozhnyi mekhanizm vliianiia adriamitsina na sintez immunoglobulinov kletkami myshinoi mielomy.

A dose-dependent stimulation of immunoglobulin synthesis by Spebr-5 cells in the presence of adriamycin is shown. This effect is significantly blocked in the presence of iron-chelating agent 2,2-bipyridine. Moreover, low doses of H2O2 and heat shock also caused enhancement of immunoglobulin synthesis. These data suggest the involvement of free radical processes in the adriamycin induced stimulation of immunoglobulin synthesis.

[Slow rhythms in the rheovasogram of the tail arteries of the unanesthetized rat]. / Medlennye ritmy v reovazogramme khvostovykh arterii nenarkotizirovannykh krys.

Parametric description of the rheovasogram (RVG) with minimal and maximal amplitude as well as the data obtained in rats suggest that the RVG slow oscillations reflect regular oscillations of vascular tone connected with the c. n. s.

[Glutamate oxalacetate and glutamate pyruvate aminotransferase activity in Streptomyces hygroscopicus 155 producing antibiotic complex]. / Gluatamat-oksaloatsetat i glytamat-piruvat aminotransferaznaia aktivnost' u Streptomyces hygroscopicus 155-produtsenta antibiotichnogo komplekca.

Antibiotic activity, aminotransferase activity and composition of intracellular amino acids in low and high potent variants of Streptomyces hygroscopicus 155 were studied. The data of the study are indicative of some general regularities characteristic of amino acid metabolism in actinomycetes: an increased pool of the main aminodonor amino acids (alanine, asparaginic and glutaminic acids) and an increased activity of aminotransferases in the cultures of both the variants when grown in media with readily assimilable nitrogen sources.

[Calcium ions and the differentiation of Streptomyces hygroscopicus 155, a producer of an antibiotic complex]. / Iony kal'tsiia i differentsiatsiia Streptomyces hygroscopicus 155, produtsenta antibioticheskogo kompleksa.

The effect of Ca2+ on differentiation of Streptomyces hygroscopicus 155 and its inactive variant 155-0 was studied. Addition of Ca2+ to the medium induced formation of the aerial mycelium in the inactive variant and accelerated formation of the aerial mycelium in the parent strain. The inhibitory effect of EGTA, verapamil, nifedipin, chlorpromazine and dilthiazeme on the aerial mycelium formation demonstrated the physiological role of Ca2+ in the process. Addition of pandavir (nigericin) and azalomycin B, the antibiotics produced by the streptomycete, induced formation of the aerial mycelium in the inactive variant. The effect was higher in the presence of Ca2+. Streptomyces hygroscopicus 155 and its inactive variant synthesized a proteolytic complex containing metalloproteases and trypsin-like proteases. The total proteolytic activity of the inactive variant was lower than that of the parent strain. Addition of Ca2+ to the medium stimulated their proteolytic activity. The inducing action of the antibiotics produced by the parent strain on differentiation of S.hygroscopicus 155-0 and the increase of their action in the presence of Ca2+ suggested that they controlled the differentiation and that such a function of the antibiotics expressed itself through the Ca2+ signal system.

[Effect of calcium ions on the differentiation of actinomycetes]. / Vliianie ionov kal'tsiia na differentsiatsiiu aktinomitsetov.

The influence of Ca2+ on the formation of the aerial mycelium in 23 actinomycete strains was studied. It was shown that in concentrations up to 5 mM exogenous Ca2+ induced the formation of the aerial mycelium in 11 out of the 23 actinomycete strains tested. The Ca2+ channel blockers (verapamil and nifedipine), calmodulin inhibitor (chlorpromazine) and Ca2+ chelator (EGTA) inhibited the aerial mycelium formation in 3 streptomycete strains out of the 23 actinomycetes. Chlorpromazine and nifedipine proved to be the most active inhibitors when used in concentrations of 0.2 to 2.5 mumol/disk. The Ca2+ concentrations of 5 mM were found to stimulate the formation of the submerged spores in S. hygroscopicus 155. The activity of ATP synthase was stimulated in the aerial and submerged spores after the Ca2+ uptake.

[Resistance of the strain Streptomyces hygroscopicus 155 to autogenous and other antibiotics]. / Ustoichivost' shtamma Streptomyces hygroscopicus 155 k sobstvennym i k drugim antibiotikam.

Streptomyces hygroscopicus 155, a strain producing pandavir (nigericin) and azalomycin, was studied with respect to resistance to its own and some other antibiotics i.e. tetracycline, gentamicin, streptomycin, erythromycin, tylosin, thiostreptone, benzylpenicillin, monensin and salinomycin. An inactive variant of the culture was used in the control.

[Preparation and regeneration of protoplasts of the strain Streptomyces hygroscopicus 155]. / Poluchenie i regeneratsiia protoplastov shtamma Streptomyces hygroscopicus 155.

Protoplasts of Streptomyces hygroscopicus 155 producing nigericin, a polyether antibiotic, were prepared. Conditions for protoplasting and optimal concentrations of lysozyme and glycine, and the age of mycelium were studied. With the method of the experiment design, concentrations of four components: CaCl2, MgCl2, KH2PO4 and TES buffer in the regeneration medium were determined. The levels of CaCl2 and TES buffer proved to be the most important parameters.

[Effect of protoplasting on antibiotic activity and resistance in the strain Streptomyces hygroscopicus 155]. / Izuchenie vliianiia protoplastirovaniia na antibioticheskuiu aktivnost' i antibiotikorezistentnost' shtamma Streptomyces hygroscopicus 155.

The influence of protoplasting and protoplast regeneration in the presence of polyethylene glycol on antibiotic activity, components of antibiotic complexes and antibiotic resistance in Streptomyces hygroscopicus 155 was studied. It was shown that the protoplasting and protoplast regeneration influenced the antibiotic activity. The protoplast fusion resulted in increased isolation of variants with higher antibiotic activity. The processes also affected the components of the antibiotic complexes but had no effect on the strain resistance to some antibiotics.

[Calcium ions in the life cycle of Streptomyces albogriseolus 444]. / Iony kal'tsiia v zhiznennom tsikle Streptomyces albogriseolus 444.

The influence of Ca2+ on the growth, antibiotic production and differentiation of Streptomyces albogriseolus 444 was studied. Consumption of the calcium ions by the strain was followed up. It was shown that Ca2+ changed the dynamics of the biomass accumulation and had no significant effect on the antibiotic production. Calcium present in the medium was assimilated more intensively during the first 24 hours of the strain growth. The own antibiotic nigericin exogenously added to the medium increased the calcium assimilation. In the presence of Ca2+ the nigericin stimulation of the strain differentiation was higher.