Occurrence of New Polyenoic Very Long Chain Acyl Residues in Lipids from Acanthamoeba castellanii

The cellular fatty acid composition of Acanthamoeba castellanii, a unicellular bacteriovorous organism, was reinvestigated. Lipids from amoebae grown axenically in proteose peptone-yeast extract-glucose medium were extracted with chloroform–methanol and separated by silicic acid column chromatography into non-polar and polar fractions. The fatty acid composition of the lipids and the double-bond position of the unsaturated acids have been determined by capillary gas chromatography-mass spectrometry (GC-MS) of their corresponding methyl esters, 2-alkenyl-4,4-dimethyloxazoline (DMOX) derivatives and dimethyldisulfide (DMDS) adducts. Evidence is given that lipids from A. castellanii in addition to the three already identified saturated straight chain fatty acids: tetradecanoic (C14:0), hexadecanoic (C16:0), octadecanoic (C18:0), and six preponderant unsaturated fatty acids: hexadecenoic (C16:1 Δ7), octadecenoic (C18:1 Δ9), octadecadienoic (C18:2 Δ9,12), eicosadienoic (C20:2 Δ11,14), eicosatrienoic (C20:3 Δ8,11,14), and eicosatetraenoic (C20:4 Δ5,8,11,14), contain additionally four very long chain unsaturated fatty acids: octacosenoic (C28:1 Δ21), octacosadienoic (C28:2 Δ5,21), triacontadienoic (30:2 Δ21,24), and triacontatrienoic (C30:3 Δ5,21,24) previously unreported in lipids of A. castellanii. These new long chain fatty acids account for approximately 25% of total fatty acids. To our knowledge, this is the first report of very long chain polyenoic fatty acids present in lipids extracted from A. castellanii cells

Selection of Endophytic Strains for Enhanced Bacteria-Assisted Phytoremediation of Organic Pollutants Posing a Public Health Hazard

Anthropogenic activities generate a high quantity of organic pollutants, which have an impact on human health and cause adverse environmental effects. Monitoring of many hazardous contaminations is subject to legal regulations, but some substances such as therapeutic agents, personal care products, hormones, and derivatives of common organic compounds are currently not included in these regulations. Classical methods of removal of organic pollutants involve economically challenging processes. In this regard, remediation with biological agents can be an alternative. For in situ decontamination, the plant-based approach called phytoremediation can be used. However, the main disadvantages of this method are the limited accumulation capacity of plants, sensitivity to the action of high concentrations of hazardous pollutants, and no possibility of using pollutants for growth. To overcome these drawbacks and additionally increase the efficiency of the process, an integrated technology of bacteria-assisted phytoremediation is being used recently. For the system to work, it is necessary to properly select partners, especially endophytes for specific plants, based on the knowledge of their metabolic abilities and plant colonization capacity. The best approach that allows broad recognition of all relationships occurring in a complex community of endophytic bacteria and its variability under the influence of various factors can be obtained using culture-independent techniques. However, for practical application, culture-based techniques have priority

Antioxidant Properties of Wafers with Added Pumpkin Seed Flour Subjected to In Vitro Digestion

In this study, our research aim was to assess the influence of pumpkin seed flour addition on the antioxidant properties, consumer acceptability, functional properties, and texture of wafers. The in vitro gastrointestinal digestion process was used to assess the effectiveness of fortification in terms of the potential bioavailability of phenolic compounds and peptides. The antioxidant activity of the obtained hydrolysates and potentially bioavailable fractions (≤3.5 kDa) was tested. The highest antiradical activity and Fe2+ chelation ability (IC50) were noted for the fraction obtained from wafers with the greatest addition of pumpkin seed flour—Pf4 (0.49 mg/mL for ABTS+*, 3.84 mg/mL for DPPH*, and 2.04 mg/mL for Fe2+ chelation). The addition of pumpkin seed flour caused the color of the wafers to change to a darker one (24.46% differences in L* between C and P4), which influenced consumer ratings. This study shows that adding pumpkin seed flour increases the peptide and phenolic contents of wafers (1.13 mg/mL and 1.01 mg/mL of peptides and 0.429 mg/mL and 0.351 mg/mL of phenolics for P4 and C hydrolysates, respectively) and enhances their antioxidant activity, with only minimal effects on taste, aroma, crispness, water and fat adsorption capacity, and foaming ability. Fractions ≤ 3.5 kDa showed greater antioxidative activity than hydrolysates, and the addition of pumpkin seed flour improved these properties. To sum up, pumpkin seeds are a valuable source of antioxidant compounds (phenolic compounds and peptides) and can be used to enrich various products

A Mutation in the <i>Mesorhizobium loti oatB</i> Gene Alters the Physicochemical Properties of the Bacterial Cell Wall and Reduces Survival inside <i>Acanthamoeba castellanii</i>

In our previous report, we had shown that the free-living amoeba Acanthamoeba castellanii influenced the abundance, competiveness, and virulence of Mesorhizobium loti NZP2213, the microsymbiont of agriculturally important plants of the genus Lotus. The molecular basis of this phenomenon; however, had not been explored. In the present study, we demonstrated that oatB, the O-acetyltransferase encoding gene located in the lipopolysaccharide (LPS) synthesis cluster of M. loti, was responsible for maintaining the protective capacity of the bacterial cell envelope, necessary for the bacteria to fight environmental stress and survive inside amoeba cells. Using co-culture assays combined with fluorescence and electron microscopy, we showed that an oatB mutant, unlike the parental strain, was efficiently destroyed after rapid internalization by amoebae. Sensitivity and permeability studies of the oatB mutant, together with topography and nanomechanical investigations with the use of atomic force microscopy (AFM), indicated that the incomplete substitution of lipid A-core moieties with O-polysaccharide (O-PS) residues rendered the mutant more sensitive to hydrophobic compounds. Likewise, the truncated LPS moieties, rather than the lack of O-acetyl groups, made the oatB mutant susceptible to the bactericidal mechanisms (nitrosative stress and the action of lytic enzymes) of A. castellanii

Occurrence of New Polyenoic Very Long Chain Acyl Residues in Lipids from Acanthamoeba castellanii

The cellular fatty acid composition of Acanthamoeba castellanii, a unicellular bacteriovorous organism, was reinvestigated. Lipids from amoebae grown axenically in proteose peptone-yeast extract-glucose medium were extracted with chloroform–methanol and separated by silicic acid column chromatography into non-polar and polar fractions. The fatty acid composition of the lipids and the double-bond position of the unsaturated acids have been determined by capillary gas chromatography-mass spectrometry (GC-MS) of their corresponding methyl esters, 2-alkenyl-4,4-dimethyloxazoline (DMOX) derivatives and dimethyldisulfide (DMDS) adducts. Evidence is given that lipids from A. castellanii in addition to the three already identified saturated straight chain fatty acids: tetradecanoic (C14:0), hexadecanoic (C16:0), octadecanoic (C18:0), and six preponderant unsaturated fatty acids: hexadecenoic (C16:1 Δ7), octadecenoic (C18:1 Δ9), octadecadienoic (C18:2 Δ9,12), eicosadienoic (C20:2 Δ11,14), eicosatrienoic (C20:3 Δ8,11,14), and eicosatetraenoic (C20:4 Δ5,8,11,14), contain additionally four very long chain unsaturated fatty acids: octacosenoic (C28:1 Δ21), octacosadienoic (C28:2 Δ5,21), triacontadienoic (30:2 Δ21,24), and triacontatrienoic (C30:3 Δ5,21,24) previously unreported in lipids of A. castellanii. These new long chain fatty acids account for approximately 25% of total fatty acids. To our knowledge, this is the first report of very long chain polyenoic fatty acids present in lipids extracted from A. castellanii cells

Assessment of Phenanthrene Degradation Potential by Plant-Growth-Promoting Endophytic Strain <i>Pseudomonas chlororaphis</i> 23aP Isolated from <i>Chamaecytisus albus</i> (Hacq.) Rothm.

Polycyclic aromatic hydrocarbons (PAHs) are common xenobiotics that are detrimental to the environment and human health. Bacterial endophytes, having the capacity to degrade PAHs, and plant growth promotion (PGP) may facilitate their biodegradation. In this study, phenanthrene (PHE) utilization of a newly isolated PGP endophytic strain of Pseudomonas chlororaphis 23aP and factors affecting the process were evaluated. The data obtained showed that strain 23aP utilized PHE in a wide range of concentrations (6–100 ppm). Ethyl-acetate-extractable metabolites obtained from the PHE-enriched cultures were analyzed by gas chromatography–mass spectrometry (GC-MS) and thin-layer chromatography (HPTLC). The analysis identified phthalic acid, 3-(1-naphthyl)allyl alcohol, 2-hydroxybenzalpyruvic acid, α-naphthol, and 2-phenylbenzaldehyde, and allowed us to propose that the PHE degradation pathway of strain 23aP is initiated at the 1,2-, 3,4-carbon positions, while the 9,10-C pathway starts with non-enzymatic oxidation and is continued by the downstream phthalic pathway. Moreover, the production of the biosurfactants, mono- (Rha-C8-C8, Rha-C10-C8:1, Rha-C12:2-C10, and Rha-C12:1-C12:1) and dirhamnolipids (Rha-Rha-C8-C10), was confirmed using direct injection–electrospray ionization–mass spectrometry (DI-ESI-MS) technique. Changes in the bacterial surface cell properties in the presence of PHE of increased hydrophobicity were assessed with the microbial adhesion to hydrocarbons (MATH) assay. Altogether, this suggests the strain 23aP might be used in bioaugmentation—a biological method supporting the removal of pollutants from contaminated environments

A Unique Sugar l-Perosamine (4-Amino-4,6-dideoxy-l-mannose) Is a Compound Building Two O-Chain Polysaccharides in the Lipopolysaccharide of Aeromonas hydrophila Strain JCM 3968, Serogroup O6

Lipopolysaccharide (LPS) is the major glycolipid and virulence factor of Gram-negative bacteria, including Aeromonas spp. The O-specific polysaccharide (O-PS, O-chain, O-antigen), i.e., the surface-exposed part of LPS, which is a hetero- or homopolysaccharide, determines the serospecificity of bacterial strains. Here, chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy techniques were employed to study the O-PS of Aeromonas hydrophila strain JCM 3968, serogroup O6. MALDI-TOF mass spectrometry revealed that the LPS of A. hydrophila JCM 3968 has a hexaacylated lipid A with conserved architecture of the backbone and a core oligosaccharide composed of Hep6Hex1HexN1HexNAc1Kdo1P1. To liberate the O-antigen, LPS was subjected to mild acid hydrolysis followed by gel-permeation-chromatography and revealed two O-polysaccharides that were found to contain a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc), which may further determine the specificity of the serogroup. The first O-polysaccharide (O-PS1) was built up of trisaccharide repeating units composed of one &alpha;-d-GalpNAc and two &alpha;-l-Rhap4NAc residues, whereas the other one, O-PS2, is an &alpha;1&rarr;2 linked homopolymer of l-Rhap4NAc. The following structures of the O-polysaccharides were established: O-PS1 &rarr;3)-&alpha;-l-Rhap4NAc-(1&rarr;4)-&alpha;-d-GalpNAc-(1&rarr;3)-&alpha;-l-Rhap4NAc-(1&rarr; O-PS2 &rarr;2)-&alpha;-l-Rhap4NAc-(1&rarr; The present paper is the first work that reveals the occurrence of perosamine in the l-configuration as a component of bacterial O-chain polysaccharides

Characterization of Active Compounds of Different Garlic (Allium sativum L.) Cultivars

Garlic (Allium sativum L.) has a reputation as a therapeutic agent for many different diseases such as microbial infections, hypertension, hypercholesterolaemia, diabetes, atherosclerosis and cancer. Health benefits of garlic depend on its content of biologically-active compounds, which differs between cultivars and geographical regions. The aim of this study was to evaluate and compare the biological activity of aqueous extracts from nine garlic varieties from different countries (Poland, Spain, China, Portugal, Burma, Thailand and Uzbekistan). Antioxidant properties were evaluated through free radical scavenging (DPPH•, ABTS•+) and ion chelation (Fe2+, Cu2+) activities. The cytotoxicity of garlic extracts was evaluated in vitro using Neutral Red Uptake assay in normal human skin fibroblasts. The obtained results revealed that garlic extracts contained the highest amount of syringic and p-hydroxybenzoic acids derivatives. The lowest IC50 values for DPPH•, ABTS•+ scavenging and Cu2+ chelating ability were determined in Chinese garlic extracts (4.63, 0.43 and 14.90 μg/mL, respectively). Extracts from Spanish cultivar Morado and Chinese garlic were highly cytotoxic to human skin fibroblasts as they reduced cellular proliferation by 70–90%. We showed diverse contents of proteins and phenolic components in garlic bulbs from different varieties. The obtained results could help to choose the cultivars of garlic which contain significant amounts of active compounds, have important antioxidant properties and display low antiproliferative effect and/or low cytotoxicity against normal human skin fibroblast BJ