Teresa Urbanik-Sypniewska

Micromolar concentration of kynurenic acid in rat small intestine

Summary.Kynurenic acid is an antagonist of glutamate and alpha-7 nicotinic acetylcholine receptors and an agonist of the g-protein-coupled receptor GPR35, which is predominantly expressed in immune and gastrointestinal tissues. In this study, we report that kynurenic acid is present in the lumen of rat small intestine in micromolar concentration sufficient to affect the GPR35 receptor. Moreover, we show that kynurenic acid can be produced by Escherichia coli. We suggest that kynurenic acid may modulate gastrointestinal function and integrity.

Two Types of Nodules Induced on Trifolium pratense by Mutants of Rhizobium leguminosarum bv. trifolii deficient in Exopolysaccharide Production

Summary Several Tn5 mutants of the symbiotic bacterium Rhizobium leguminosarum bv. trifolii 24.1 that fail to synthesize acidic exopolysaccharide, are able to induce non-nitrogen-fixing nodules on Trifolium pratense . Hybridization analysis indicated that Tn5 insertions were located on megaplasmids pRtb and pRtc and on the chromosome of R. leguminosarum bv. trifolii 24.1. Microscopic analysis of nodules induced by these non-mucoid mutants revealed two types of nodules: infected nodules with infection threads and bacteroids, and non-infected nodules which neither contained infection threads nor bacteroids. Ultrastructural data on bacteroid development and plant cell differentiation in nodules induced by the mutants indicate a decrease in bacteroid and plant metabolism. Starch accumulation in the plant plastids and presence of osmiophilic droplets in the plant cells of the peripheral tissue was observed.

Changes in acyl lipid and fatty acid composition in thylakoids of copper non-tolerant spinach exposed to excess copper

Summary The effect of toxic concentrations of Cu on acyl lipid and fatty acid composition in thylakoid membranes of non-tolerant spinach plants exposed to excess Cu was investigated. In Cu-treated plants a decrease in acyl lipid content and changes in its molar distribution were found. Thylakoid lipid degradation was accompanied by changes in fatty acid composition, especially that of 18:3, 16:1 and 16: 0. The obtained results are discussed in terms of the indirect effect of long term action of excess Cu on the thylakoid membrane structure and decrease of its photochemical activities.

Galactolipase Activity of Chloroplasts in Cadmium-treated Runner Bean Plants

Summary The galactolipase activity in chloroplasts of primary leaves of runner bean plants ( Phaseolus coccineus L., cv. Piekny Jaś) grown in Cd-containing nutrient solution was determined and compared with that obtained from plants developed under normal nutrient conditions. A several times higher galactolipase activity was found in Cd-treated plants in comparison with controls. A high galactolipase activity was accompanied by a decrease in all acyl lipid classes (mainly MGDG), components of thylakoid membranes and FFA release. Chloroplasts of Cd-treated plants revealed a lower photosynthetic oxygen evolution than control plants. Decrease of this activity was considerably eliminated in the presence of BSA, used in the chloroplast isolation procedure. In in vitro experiments, inhibition of galactolipase activity due to Cd concentration in the incubation mixture was found. The obtained results are discussed in terms of the indirect effect of long term action of Cd on the decrease of photochemical activity of thylakoids related to thylakoid bound galactolipase activity.

Rhizobium leguminosarum bv. trifolii PssP Protein Is Required for Exopolysaccharide Biosynthesis and Polymerization

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS) that is important for the induction of nitrogen-fixing nodules on clover. Recently, three genes, pssN, pssO, and pssP, possibly involved in EPS biosynthesis and polymerization were identified. The predicted protein product of the pssP gene shows a significant sequence similarity to other proteins belonging to the PCP2a family that are involved in the synthesis of high-molecular-weight EPS. An R. leguminosarum bv. trifolii TA1 mutant with the entire coding region of pssP deleted did not produce the EPS. A pssP mutant with the 5 end of the gene disrupted produced exclusively low-molecular-weight EPS. A mutant that synthesized a functional N-terminal periplasmic domain but lacked the C-terminal part of PssP produced significantly reduced amounts of EPS with a slightly changed low to high molecular form ratio. Mutants affected in the PssP protein carrying a stable plasmid with a constitutively expressed gusA gene induced nodules on red clover that were not fully occupied by bacteria. A mutant with the entire pssP gene deleted infected only a few plant cells in the nodule. The pssP promoter-gusA reporter fusion was active in bacteroids during nodule development.

Mutation in the pssB-pssA intergenic region of Rhizobium leguminosarum bv. trifolii affects the surface polysaccharides synthesis and nitrogen fixation ability

Summary A Rhizobium leguminosarum bv. trifolii Tn 5 transposon mutant deficient in exopolysaccharide biosynthesis was found to form non-nitrogen fixing nodules on clover. Root nodules induced by the mutant contained aberrant infection threads and few bacteroids. Sequence analysis of the transposon insertion site localized the mutation in the pssB-pssA intergenic region affecting the exopolysaccharide biosynthesis. The mutant also showed decreased sensitivity to SDS and deoxycholate and displayed a changed lipopolysaccharide (LPS) banding pattern compared to the wild-type strain TA1. The alteration in the O-polysaccharide part of LPS was confirmed by Western immunoblotting with polyclonal antibodies. LPS preparations of strain TA1 and the mutant strain only reacted with their homologous sera. The common epitopes in LPS from bacteroids and free-living rhizobia were revealed by immunogold assay. The results of this study indicate that the pssB-pssA region of R. leguminosarum bv. trifolii is important for the polysaccharide synthesis.

Effect of authentic flavonoids and the exudate of clover roots on growth rate and inducing ability of nod genes of Rhizobium leguminosarum bv. trifolii

Abstract A number of authentic flavonoids and exudate of sprouted seeds of Trifolium pratense were used to test the growth rate and nodC:-:lacZ expression controlled by nodD gene in Rhizobium leguminosarum bv. trifolii strains 24.1 and 288. All commercial flavonoids and clover exudate increased the growth rate of fresh field isolate 288 in defined minimal medium. Apigenin and exudate inhibited the growth of laboratory strain 24.1. There was no correlation between the inducing activity and the growth rate of R. leguminosarum bv. trifolii strains. Unsubstituted flavone, quercetin, hesperetin and hesperidin were not active in the induction of nodC-gene expression. Biological activity of aglycones and glycosides depended on their chemical structure i.e. quercetin-3-O-glycoside was more active than its aglycone, but hesperetin-7-O-glycoside was not active similarly to hesperetin. These results suggest that the presence of the mixture of flavonoids in the exudate of the germinating seeds of clover can influence rhizobial population. Biological activity of flavonoids, measured by level of induction of β-galactosidase, depended on the structure of the compounds and also rhizobial strain tested.

Occurrence and Taxonomic Significance of Oxo-fatty Acids in Lipopolysaccharides from Members of Mesorhizobium

Lipopolysaccharides (LPSs) isolated from seven strains of Mesorhizobium were studied for the presence of fatty acids with particular attention for 27-oxooctacosanoic acid and 4-oxo fatty acids. The LPSs from all analysed strains contained various amounts of 27-oxo-28:0 and all of them, with the exception of Mesorhizobium tianshanense, contained also 4-oxo fatty acids (4-oxo-20:0, 4-oxo-i-21:0, 4-oxo-22:0). The group of amide-linked fatty acids consisted of a wide range of 3-hydroxylated and 4-oxo fatty acids whereas all the nonpolar as well as the (omega-1) hydroxylated long-chain acids and the 27-oxo-28:0 fatty acids were ester-linked. The characteristic spectrum of 3-hydroxy fatty acids and presence of 27-OH-28:0 as well as 27-oxo-28:0 acid in LPSs of Mesorhizobium showed that these strains were closely related. Therefore the lipid A fatty acid pattern could be a useful chemotaxonomic marker which helps to isolate the Mesorhizobium group from rhizobium bacteria during the classification process.

Effect of kynurenic acid on the viability of probiotics in vitro.

Probiotics are bacteria that are commercially available as dietary supplements. One of the important properties of probiotics is their ability to survive in the intestine. Recent evidence has identified kynurenic acid (KYNA) as a bactericidal constituent of intestinal fluid. These data led us to study the influence of KYNA on the viability of selected probiotics. We found that KYNA supported the growth of bacteria in the probiotics Acidolac (Lactobacillus acidophilus, Bifidobacterium) and Lakcid Forte (Lactobacillus rhamnosus) or retarded the growth of bacteria from the Acidolac, BioGaia (Lactobacillus reuteri Protectis), Dicoflor (Lactobacillus rhamnosus GG), Lacium (Lactobacillus plantarum) and Trilac (Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium animalis subsp. lactis) probiotics depending on its concentration. KYNA did not affect the viability of bacteria from the probiotic Linex (Lactobacillus acidophilus LA-5, Bifidobacterium animalis subsp. lactis BB-12). Our results suggest a potential role of KYNA in the regulation of bacterial growth in the digestive system.

Do Ca2+-chelating polysaccharides reduce calcium ion release from gypsum-based biomaterials?

BackgroundCalcium sulphate, a widely used bone filler, may negatively affect human osteoblasts due to release of high quantities of calcium ions. To reduce this effect, an attempt was made to enrich calcium sulphate with Ca2+-chelating plant and rhizobial exopolysaccharides (EPS).MethodologyIncubation of polysaccharide-enriched calcium sulphate composites was performed in DMEM/F12 medium. Ca2+ (and Mg2+ and Pi) levels were estimated using standardised, spectrophotometry-based kits. Composite surface morphology was tested using SEM technique.ResultsRhizobial EPS was found slightly less effective at Ca2+ chelation than sodium alginate. Both polysaccharides may be used as gypsum supplements in the form of setting liquids (0.3% total mass), but only sodium alginate may be used as a powder (up to 5% total mass of the composite). Polysaccharide-triggered reduction of Ca2+ release reached the level of 50% during the first 2.5 h of incubation, then decreased significantly.ConclusionsBoth tested polysaccharides possess calcium-chelating properties. However, although alginate caused a reduction in Ca2+ levels in the media incubated with the gypsum samples, the reduction was too short lived to provide a long-term effect. Further modification of the composite content using calcium-deficient hydroxyapatite and low-molecular weight rhizobial EPS with higher solubility could bring more satisfactory results.