Andrea Bilková

An amine oxidase in seedlings of Papaver somniferum L.

Amine oxidase (AO) from 4-d-old seedlings of Papaver somniferum L. (Papaveraceae) was purified (58-fold) by using ammonium sulphate precipitation and chromatography on Sephadex G-150 and HA-Ultrogel columns. The most readily oxidized substrate was tyramine and other aromatic amines, while aliphatic amines cadaverine and putrescine were oxidized more slowly. Cu chelating and carbonyl reagents are the most effective inhibitors of poppy amine oxidase. Immunoblotting analysis showed cross reactivity of AO protein from poppy seedlings with polyclonal antisera against AO from pea. Obtained Mr value for AO from poppy (83 kDa) corresponds to that of copper AOs (75 – 90 kDa). These results suggest that the amine oxidase from poppy seedlings is a copper containing and tyramine specific AO.

Antimicrobial Activity and Urease Inhibition of Schiff Bases Derived from Isoniazid and Fluorinated Benzaldehydes and of Their Copper(II) Complexes

In order to evaluate the influence of substitution on biological properties of Schiff bases and their metal complexes, a series of differently substituted fluorine-containing Schiff bases starting from the drug isoniazid (isonicotinylhydrazide) were prepared and their structures were established by single-crystal X-ray diffraction. Also, four copper(II) complexes of these Schiff bases were synthesized. The prepared compounds were evaluated for their antimicrobial activity and urease inhibition. Two of the Schiff bases exerted activity against C. albicans. All copper(II) complexes showed excellent inhibitory properties against jack bean urease, considerably better than that of the standard inhibitor acetohydroxamic acid.

Isolation and identification of new lactobacilli from goatling stomach and investigation of reuterin production in Lactobacillus reuteri strains

Five new strains of lactobacilli isolated from goatling’s stomach were identified by molecular–biological approaches. Profiles of fermentable saccharides, Gram staining, and cell morphology were also determined. They were identified as Lactobacillus reuteri (strains KO4b, KO4m, KO5) and as Lactobacillus plantarum (strains KG1z, KG4). In DNA samples of all newly isolated L. reuteri strains as well as in L. reuteri E (Lreu E; originated from lamb), the part of gldC gene, coding large subunit of glycerol dehydratase, that is necessary for 3-hydroxypropionaldehyde (3-HPA; reuterin) production, was amplified using two designed primer sets. However, the 3-HPA production was revealed only in the strain Lreu E. It produced five- or ten-fold lower amount of 3-HPA in comparison with probiotic L. reuteri ATCC 55730 in aerobic or anaerobic conditions, respectively. Moreover, Lreu E completely lost its production ability after ca. five passages in MRS medium. The co-incubation of Lreu E, but not other L. reuteri isolates, with Escherichia coli re-induced 3-HPA production. In the case of L. reuteri ATCC 55730, the 3-HPA production increased more than four times after co-incubation with E. coli.

Identification and biological activity of potential probiotic bacterium isolated from the stomach mucus of breast-fed lamb

The lactic acid bacterium E isolated from the stomach mucus of breast-fed lamb was identified by sequencing of 16S rDNA fragment and species-specific PCR as Lactobacillus reuteri. Its potential antimicrobial activity and ability to modulate immune system in vitro and in vivo was determined. The growth inhibition of potential pathogens decreased from Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica ser. Minnesota to Escherichia coli. The lowest inhibition activity was observed in the case of Candida albicans. The ability of L. reuteri E to modulate biological activities of human and mouse mononuclear cells was estimated in vitro and in vivo, respectively. The production of IL-1β by monocytes in vitro was significantly induced by L. reuteri E (relative activity 2.47). The ability to modulate biological activities of mononuclear cells by living L. reuteri E cells in vitro in comparison to disintegrated L. reuteri E cells in vivo differed. For example lysozyme activity in vitro was inhibited while in vivo was stimulated (relative activities 0.30 and 1.83, respectively). The peroxidase activity in vitro was stimulated while in vivo was inhibited (relative activities 1.53 and 0.17, respectively). Obtained results indicate that L. reuteri E is potential candidate to be used in probiotic preparations for animals and/or human.

Characterization of Polyphenol Oxidase from the Latex of Opium Poppy

Polyphenol oxidase from the latex of opium poppy was purified to the electrophoretic homogeneity by affinity chromatography using p-aminobenzoic acid as a ligand coupled to Sepharose CL-4B by divinyl sulphone activation method. The purified enzyme was used to prepare the polyclonal antibodies. The purified latex PPO exhibited high diphenolase activity in comparison with almost unmeassurable monophenolase activity. Both of these activities were sensitive to the activation with sodium dodecyl sulphate. Two isoforms (65 and 40 kDa) of latex PPO were separated by the gel filtration. There were no differences in substrate specifity (weak monophenolase and high diphenolase activity) and sensitivity to inhibitors between these isoforms, but they showed differences in electrophoretic mobility.

Antimicrobial susceptibility and immunomodulatory properties of lamb isolate of lactobacillus mucosae, new probiotic candidate

Abstract In the process of selecting a new probiotic candidate, several bacteria were isolated from the stomach mucosa of a lamb. Among them, three lactobacilli strains were identified and partially characterised. The strain, Lactobacillus mucosae D, showed several characteristics appropriate to the probiotics. In this study, we have focused on the further characterisation of L. mucosae D and testing of its ability to modulate metabolic and immunomodulatory activities of human mononuclear cells in vitro. L. mucosae D is resistant to antibiotics, like penicillin G, oxacillin, vancomycin and chemotherapeutics ofloxacin and ciprofloxacin. In in vitro conditions, L. mucosae D caused a significant increase in phagocytic activity and index (relative activities 1.05 and 1.44, respectively) of human monocytes. It decreased bactericidal activities of monocytes against Escherichia coli (relative activity 0.73) and Staphylococcus aureus (relative activity 0.36), whereas, candidacidal activity was enhanced (relative activity 1.15). Metabolic activities, lysozyme and peroxidase activity, of mononuclear cells were not changed or increased, respectively. L. mucosae D displayed the ability to enhance production of pro-inflammatory cytokine, IL-1β, in monocytes in vitro (relative activity 2.60). Therefore, we state that lamb isolate, L. mucosae D, has the required attributes for being a potential probiotic candidate. Slovak abstract S cieľom ziskať novych probiotickych kandidatov, bolo zo žaludočnej sliznice jahňaťa izolovanych niekoľko bakterii. Tri z nich boli identifikovane ako laktobacily a boli čiastočne charakterizovane. Kmeň Lactobacillus mucosae D preukazal v predošlych experimentoch niekoľko vlastnosti typickych pre probiotika. V tejto praci sme sa zamerali na ďalšiu charakterizaciu Lactobacillus mucosae Da sledovanie jeho schopnosti modulovať metabolicke a imunomodulačne vlastnosti ľudskych mononukleovych buniek v podmienkach in vitro. Zistili sme, že L. mucosae D je rezistentny voči nasledujucim antibiotikam: penicilin G, oxacilin, vankomycin a chemoterapeutikam ofloxacin a ciprofloxacin. V podmienkach in vitro L. mucosae D sposobil signifikantne zvyšenie fagocytovej aktivity a indexu (relativne aktivity 1,05 a 1,44) ľudskych monocytov. Baktericidnu aktivitu monocytov voči Escherichia coli znižil (relativna aktivita 0,73) rovnako ako aktivitu voči Staphylococcus aureus (relativna aktivita 0,36), zatiaľ čo kandidacidnu aktivitu zvyšil (relativna aktivita 1,15). Z metabolickych aktivit mononukleovych buniek sme u lyzozymovej aktivity nepozorovali žiadnu zmenu, naopak peroxidazova aktivita bola zvyšena. L. mucosae D preukazal schopnosť potencovať produkciu prozapaloveho cytokinu IL-1β monocytmi v podmienkach in vitro (relativna aktivita 2,60). Na zaklade ziskanych vysledkov považujeme jahňaci izolat L. mucosae D za potencialne probioticky.