Martina Dubničková

Bilayer thickness and lipid interface area in unilamellar extruded 1,2-diacylphosphatidylcholine liposomes: a small-angle neutron scattering study.

Small-angle neutron scattering (SANS) experiments have been performed on large unilamellar liposomes prepared from 1,2-dilauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-phosphatidylcholine (DMPC) and 1,2-distearoylphosphatidylcholine (DSPC) in heavy water by extrusion through polycarbonate filters with 500 A pores. The neutron scattering intensity I(Q) in the region of scattering vectors Q corresponding to 0.0015 A(-2) < or = Q(2) < or = 0.0115 A(-2) was fitted using a step function model of bilayer neutron scattering length density and supposing that the liposomes are spherical and have a Gaussian distribution of radii. Using the lipid volumetric data, and supposing that the thickness of bilayer polar region equals to d(H) = 9+/-1 A and the water molecular volume intercalated in the bilayer polar region is the same as in the aqueous bulk aqueous phase, the steric bilayer thickness d(L), the lipid surface area A(L) and the number of water molecules per lipid molecule N intercalated in the bilayer polar region were obtained: d(L) = 41.58+/-1.93 A, A(L) = 57.18+/-1.00 A(2) and N = 6.53+/-1.93 in DLPC at 20 degrees C, d(L) = 44.26+/-1.42 A, A(L) = 60.01+/-0.75 A(2) and N = 7.37+/-1.94 in DMPC at 36 degrees C, and d(L) = 49.77+/-1.52 A, A(L) = 64.78+/-0.46 A(2) and N = 8.67+/-1.97 in DSPC at 60 degrees C. After correcting for area thermal expansivity alpha approximately 0.00417 K(-1), the lipid surface area shows a decrease with the lipid acyl chain length at 60 degrees C: A(L) = 67.56+/-1.18 A(2) in DLPC, A(L) = 66.33+/-0.83 A(2) in DMPC and A(L) = 64.78+/-0.46 A(2) in DSPC. It is also shown that a joint evaluation of SANS and small-angle X-ray scattering on unilamellar liposomes can be used to obtain the value of d(H) and the distance of the lipid phosphate group from the bilayer hydrocarbon region d(H1).

Dynamic light scattering and electrokinetic potential of bis(quarternary ammonium bromide) surfactant micelles as the function of the alkyl chain length

Abstract The micellar properties of bis(quarternary ammonium bromide) surfactants were studied by means of dynamic light scattering and electrokinetic measurements. The coexistence of small spherical micelles and large aggregates for surfactants with eight to 12 carbon atoms in the alkyl chain is observed. Below this region, only spherical micelles appear and above 12 carbon atoms, large aggregates are present. The electrokinetic measurements of zeta potential correspond with the light scattering results. Bimodality in the peaks was observed in the region of nine to 12 carbon atoms in the alkyl chain. As follows from the light scattering results, solubilization with a monomeric surfactant showed that, in the case of dodecyltrimethylammonium bromide (DTAB) and 12-4-12 surfactant, the presence of large aggregates decreases strongly with increasing DTAB concentration in the system. No such observation was made when hexadecyltrimethylammonium bromide (CTAB) was present in the system with 16-4-16 surfactant, where only unimodal size spectra were registered.

Adaptive changes in fatty acids ofE. coli strains exposed to a quaternary ammonium salt and an amine oxide

Resistant strains ofEscherichia coli were obtained by stepwise cultivation in media with increasing concentration of antimicrobially active 1-(methyldodecyl)dimethylamine oxide and 1-(methyldodecyl)trimethylammonium bromide. Adaptive changes were determined in the fatty-acid (FA) composition in an isolated lipopolysaccharide sample from the outer membrane of these strains. The composition of this FA mixture from adapted strains was compared with that of FA from a sensitive strain. The differences were found in level of palmitic, heptadecanoic, heptadecenoic, heptadecadienoic and nonadecenoic acids. In addition, the adapted strains differed from each other in the content of myristic, pentadecanoic, stearic and linoleic acids.

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.

Activation of human leukocytes by lipid A from E. coli strains adapted to quaternary ammonium salt and amine oxide.

The immunomodulatory activities of monophosphoryl lipid A (MLA) and diphosphoryl lipid A analogues obtained from the sensitive strain ofE. coli and from the resistant strains adapted to a quaternary ammonium salt and an amine oxide were compared. All analogues considerably stimulated the activity of human leukocytes although the analogue from the sensitive strain at a higher concentration significantly suppressed phagocytosis. The MLA analogue exhibited a suppressive effect on the microbicidal activity of human leukocytes againstE. coli and the peroxidase activity. Adaptation of bacteria to amphiphilic antimicrobial compounds, which is accompanied by chemical changes in their lipid A, only slightly reduced their immunomodulatory activity when compared with the analogue from the sensitive strain. On the other hand, the diphosphoryl analogues were less active than MLA.

Analysis of antimicrobial and immunomodulatory substances produced by heterofermentative Lactobacillus reuteri

Antimicrobial and immunomodulatory potential of various Lactobacillus reuteri strains is closely connected to their metabolite production profile under given cultivation conditions. We determined the in vitro production of antimicrobial substances such as organic acids, ethanol, and reuterin by four strains of L. reuteri (L. reuteri E, L. reuteri KO5, L. reuteri CCM 3625, and L. reuteri ATCC 55730). All studied L. reuteri strains showed the ability to produce lactic acid, acetic acid, and ethanol with concominant consumption of glucose and together with phenyllactic acid—a potent antifungal compound—with concominant consumption of phenylalanine. The reuterin production from glycerol was confirmed for all analyzed lactobacilli strains except L. reuteri CCM 3625. Production of organic acids, ethanol, and reuterin is significantly involved in antimicrobial activity of lactobacilli which was determined using the dual-culture overlay diffusion method against six indicator bacteria and five indicator moulds. In comparison to the referential L. reuteri ATCC 55730, the highest inhibition potential was observed against Escherichia coli CCM 3988 and Pseudomonas aeruginosa CCM 3955. Among analyzed indicators of moulds, the growth of Alternaria alternata CCM F-128 was the most inhibited by all four analyzed L. reuteri strains. Finally, the immunomodulatory potential of analyzed lactobacilli were proven by the determination of the in vitro production of biogenic amines histamine and tyramine. L. reuteri CCM 3625 was able to produce tyramine, and L. reuteri E and L. reuteri KO5 were able to produce histamine under given cultivation conditions.

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.

Human phagocytic cell response to histamine derived from potential probiotic strains of Lactobacillus reuteri

Histamine derived from lactobacilli isolates is considered to be a potential immunomodulator able to interact with the host immune system. We tested the effect of pure histamine (0.413 mM) together with the effect of cell-culture supernatants (CCS) containing different concentration of histamine produced by two of Lactobacillus reuteri isolates on the activities of antioxidant enzyme, as well as on the phagocytic activity of human leucocytes (HL). Phagocytic activity represents the non-specific immune response of HL homogenate, in vitro. Analysed histamine-producers were represented by a goatling isolate named L. reuteri KO5 and a lamb isolate named L. reuteri E and histamine production was determined using HPLC method connected with UV detection. Concretely, the samples contained the mixture of isolated HL and the addition of lactobacilli CCS at three different final concentrations of histamine ∼ 0.1, 1.8 and 5.4 mM. It was found that pure histamine (0.413 mM) did not significantly influence the oxidant-antioxidant balance in HL demonstrated by unchanged degree of HL lipid peroxidation. However, at the same time, the final activity of catalase and superoxide dismutase were significantly changed (p ≤ 0.001). Moreover, the phagocytic index (p ≤ 0.01), lysozyme (p ≤ 0.05) and peroxidase activity (p ≤ 0.001), and production of IL-1β significantly decreased. CCS containing different concentration of produced histamine were also able to modulate the host non-specific immune response together with the enzymatic activity of SOD and catalase too. However, our findings indicated that the impact of lactobacilli histamine is strictly strain-dependent and concentration dependent. Moreover, it seems that histamine is not the only one lactobacilli metabolite, which may play an important role in overall immunomodulatory and antioxidant potential of tested lactobacilli.

The effects of lipid A on gamma-irradiated human peripheral blood lymphocytes in vitro

The modulatory effects of lipid A (diphosphoryl lipid A (DLA) and monophosphoryl lipid A (MLA)) on apoptosis induction and DNA structure damage (single and double-strand breaks (SSBs and DSBs, respectively)) in peripheral human blood lymphocytes are studied for 60Co gamma-irradiation. It is shown that in the presence of these agents the amount of apoptotic cells increases compared with the irradiated control samples. The effect is most strongly pronounced for DLA. In its presence, a significant increase is observed in the number of radiation-induced DNA SSBs and DSBs. Possible mechanisms are discussed of the modifying influence of the used agents on radiation-induced cell reactions

Effects of lipid A analogues of the strains of E. coli on complement and myeloperoxidase activation in myocardial reperfusion following ischaemia in rats

Effects of lipid A analogues of the strains of E. coli on complement and myeloperoxidase activation in myocardial reperfusion following ischaemia in rats The aim of the study was to evaluate the ability of lipid A analogues obtained from the native strain (LAS) or strains of E. coli resistant to either an amine oxide (modLANO) or to a quaternary ammonium salt (modLBr) to reduce myocardial ischaemia and reperfusion injury in rats. The 0.5, 2, 4, 24 h pretreatment of rats with lipid A from a strain of E. coli resistant to an amine oxide (modLANO 0.5 mg/kg i.v.) in an in vitro model of myocardial ischemia lasting 10 min followed by 10 min reperfusion did not reduce the myeloperoxidase (MPO) and complement activities. In an in vivo study, the 24 h pretreatment of rats with modLANO in a dose of 0.5 mg/kg i.v. only significantly decreased both the MPO and complement activities in serum (p<0.01). The analogues of lipid A indicate abilities to influence the myocardial ischaemia-reperfusion injury in times-dependent and structures-dependent ways. Vplyv analógov lipidu a kmeňov E. coli na komplementovú a myeloperoxidázovu aktiváciu počas reperfúzie po ischémii srdca potkanov Cieľom práce bolo zistiť, či analógy lipidu A získané z natívneho kmeňa (LAS), a z kmeňa E. coli rezistentného na amínoxid (modLANO) alebo na kvartérnu amóniovú soľ (modLBr) znižujú ischemicko - reperfúzne poškodenie srdca potkanov. Lipid A z rezistentného kmeňa E. coli na amínoxid (modLANO 0,5 mg/kg i.v.) neznížil v in vitro pokusoch po 10 minút trvajúcej ischémii srdca a následnej 10 min reperfúzii myeloperoxidázovú (MPO) ani komplementovú aktivitu pri rôznej dlžke predliečenia (0,5, 2, 4, 24 h) potkanov. V in vivo pokusoch 24 h predliečenie potkanov s modLANO v dávke 0,5 mg/kg i.v. významne znížilo MPO aj komplementovú aktivitu v sére (p<0,01). Analógy lipidov A prejavili schopnosť ovplyvniť poškodenie myokardu v podmienkach ischémie a reperfúzie v závislosti od času pôsobenia a ich štruktúry.