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.

Phenol-water extraction of Rhizobium loti NZP2213 cells allowed a simultaneous isolation of two structurally different lipopolysaccharides from the aqueous (LPS-W) and phenol (LPS-P) phase that differed in their sodium deoxycholate-PAGE pattern and composition. LPS-W showed a profile indicating an R-type LPS; LPS-P had a cluster of poorly resolved bands in the high-molecular-weight region. LPS-P contained large amounts of 6-deoxy-L-talose (6dTal), and a small amount of 2-O-methyl-6-deoxy-talose (molar ratio approximately 30:1), both of which were completely absent in LPS-W. Methylation analysis gave only one major product, 2,4-di-O-methyl-6dTal, indicating that the O-chain is composed of a homopolymer of 1,3-linked 6dTal, having the methylated 6dTal (2-O-Me-6dTal) probably localized at the non-reducing end of the O-chain. This homopolymeric O-chain was additionally O-acetylated, as evidenced by GC-MS and by 13C NMR analysis. The lipid A moieties of both LPS-W and LPS-P showed almost identical composition, with six different 3-OH fatty acids and with two, so far not described, long-chain 4-oxo-fatty acids, all being amide-linked, and with 27-OH-28:0 as the main ester-linked fatty acid. Lipid A was of the lipid ADAG-type, i.e., having a (phosphorylated) 2,3-diamino-2,3-dideoxy-D-glucose-containing lipid A backbone. Lipid ADAG is widespread among species of the alpha-2 group of Proteobacteria, but has so far not been encountered in any other rhizobial or agrobacterial species.

An antifungal active fraction (AAF) from the coelomic fluid (CF) of the earthworm Dendrobaena veneta was isolated. The aim of the study was to analyze the antifungal activity of the AAF and to carry out chemical characterization of the fraction. The active fraction showed antifungal activity against a clinical C. albicans isolate, C. albicans ATCC 10231, and C. krusei ATCC 6258. It effectively reduced the metabolic activity of C. albicans cells and influenced their morphology after 48 hours of incubation. Scanning electron microscopy (SEM) images revealed loss of integrity of the cell wall induced by the active fraction. Calcofluor White staining showed changes in the structure of the C. albicans cell wall induced by the AAF. The fungal cells died via apoptosis and necrosis after the treatment with the studied fraction. Electrophoresis under native conditions revealed the presence of two compounds in the AAF, while SDS/PAGE gel electrophoresis showed several protein and carbohydrate compounds. The active fraction was analyzed using Raman spectroscopy, MALDI TOF/TOF, and ESI LC-MS. The Raman analysis confirmed the presence of proteins and determined their secondary structure. The MALDI TOF/TOF analysis facilitated detection of four main compounds with a mass of 7694.9 m/z, 12292.3 m/z, 21628.3 m/z, and 42923.2 m/z in the analyzed fraction. The presence of carbohydrate compounds in the preparation was confirmed by nuclear magnetic resonance (NMR) and gas chromatography (GC-MS). The ATR-FTIR spectrum of the AAF exhibited high similarity to the spectrum of egg white lysozyme. The AAF showed no endotoxicity and cytotoxicity towards normal skin fibroblasts (HSF); therefore, it can be used for the treatment of skin and mucous membrane candidiasis in the future. Given its efficient and selective action, the fraction seems to be a promising preparation with antifungal activity against C. albicans.

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.

The potential role of currency in the spread of pathogenic microflora has been evaluated in many countries. In this study Polish paper notes and the coins in general circulation were assayed for the presence of cultivable bacteria and fungi. Bacterial isolates identification was based on cultural and biochemical characters and by comparison of the 16S rRNA gene sequence. Fungal isolates were recognized with biochemical and morphological criteria. Coagulase-negative staphylococci, (43.6% of the total bacterial count) including Staphylococcus saprophyticus, S. epidermidis, and S. hominis, and Enteroccus spp. (30.8% of the total bacterial count), i.e. E.faecalis, E.faecium and E. durans, were the most numerous bacterial contamination. Penicillium spp., and Aspergillus spp. were the most frequently detected moulds whereas Candida spp. was the most frequent yeast isolated from currency. A visible dependence between the banknote denomination, the physical condition of paper currency, and the number of bacteria and fungi was found. The overall count of bacteria isolated from currency was thousand-fold higher than that of fungal isolates. The total amount of bacteria and fungi recovered from the coins was approximately 2.7-fold lower than that isolated from the notes. In summary, the Polish currency notes were found to be contaminated mainly with commensal bacteria and fungi while the opportunistic pathogenic microorganisms Escherichia coli, Pseudomonas stutzeri and C. albicans were detected at a low frequency.

Rhizobium leguminosarum bv. trifolii pssA encodes a glucosyl-isoprenylphosphate (IP)-transferase involved in the first step of exopolysaccharide (EPS) synthesis. It was found that the pssA gene is an important target for regulation of this biosynthetic pathway. The data of this study indicate that pssA transcription is a very complex and mainly positively regulated process. A detailed analysis of a 767-bp-long pssA upstream region revealed the presence of several sequence motifs recognized by regulatory proteins that are associated with phosphate-, carbon-, and iron-dependent regulation. In addition, numerous inverted repeats of different lengths have been identified in this region. pssA transcription is directed from two distal P1 and proximal P3 promoters whose sequences demonstrate a significant identity to promoters recognized by RNA polymerase sigma factor 70 . Among rhizobial proteins, RosR seems to be a primary regulator that positively affects pssA expression. This protein binds to RosR box 1 located downstream of the P1 promoter. In addition, phosphate and the carbon source strongly affect pssA transcription. A significantly lower level of pssA expression was observed in both the wild-type strain growing under phosphate-rich conditions and the phoB mutant. In this regulation, the PhoB protein and Pho box 2 located upstream of the P3 promoter were engaged. pssA transcription is also significantly affected by glucose. Transcriptional analysis of a set of pssA-lacZ fusions expressed in Escherichia coli wild-type and cyaA and crp mutants confirmed that cyclic AMP (cAMP) receptor protein (CRP) and two cAMP-CRP boxes located upstream of the P1 are required for this upregulation. Moreover, the production of EPS was totally abolished in R. leguminosarum bv. trifolii mutant strains 4440 and 1012 containing a Tn5 insertion downstream of the P3 promoter and downstream of the P3 ؊35 hexamer, respectively. R hizobium leguminosarum bv. trifolii is a soil bacterium able to induce formation of nitrogen-fixing root nodules on its host plant, clover (Trifolium pratense). The establishment of symbiosis is a complex process involving an exchange of a series of signals between the plant and the bacterium; among them, flavonoids secreted from plant roots and bacterial Nod factors are the best characterized signals (1). In addition, acidic exopolysaccharide (EPS) secreted by rhizobia in large amounts plays a crucial role in the symbiotic interactions with legumes that form indeterminatetype nodules (e.g., Trifolium, Pisum, Vicia, and Medicago) (2). EPS-deficient mutants of R. leguminosarum bv. trifolii induce formation of small, only partially infected nodules on clover plants that are ineffective in nitrogen fixation (3-5). In free-living cells of rhizobia, EPS has several other functions such as nutrient gathering, protection against environmental stresses, attachment to abiotic and biotic surfaces, and biofilm formation, ensuring adaptation of these bacteria to changing environmental conditions (6). EPS produced by R. legu...

It is known that earthworm coelomic fluid (CF) can affect not only cancer but also normal cells. The study demonstrated that the CF of the earthworm Dendrobaena veneta exhibited cytotoxicity against A549 lung cancer cells but did not toward the bronchial epithelial cell line BEAS‐2B. The selective effect on the tumor cells was achieved after a short‐term CF heat pre‐treatment at 70 °C. The cytotoxic effect of the CF was time‐ and concentration‐dependent. The CF noticeably decreased the viability and affected the morphology of the A549 cells. Scanning electron microscopy revealed a different degree of destruction of the nucleus and cytoplasm of A549 cells. As determined by atomic force microscopy, the cell surface roughness increased while the cell stiffness was reduced upon the CF treatment. A twofold increase in the caspase 3, 4, 5, and 10 levels was observed in the A549 cells after the incubation with the CF. The results obtained by flow cytometry using Annexin V confirmed the proapoptotic effect of the earthworm CF on A549 lung cancer cells. The D. veneta CF and active fraction obtained with cytotoxicity toward A549 lung cancer is an interesting and promising preparation for further biological, chemical, and biomedical research.

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