Multimodal Spectroscopic Imaging of Pea Root Nodules to Assess the Nitrogen Fixation in the Presence of Biofertilizer Based on Nod-Factors.

Multimodal spectroscopic imaging methods such as Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI), Fourier Transform Infrared spectroscopy (FT-IR) and Raman spectroscopy were used to monitor the changes in distribution and to determine semi quantitatively selected metabolites involved in nitrogen fixation in pea root nodules. These approaches were used to evaluate the effectiveness of nitrogen fixation by pea plants treated with biofertilizer preparations containing Nod factors. To assess the effectiveness of biofertilizer, the fresh and dry masses of plants were determined. The biofertilizer was shown to be effective in enhancing the growth of the pea plants. In case of metabolic changes, the biofertilizer caused a change in the apparent distribution of the leghaemoglobin from the edges of the nodule to its centre (the active zone of nodule). Moreover, the enhanced nitrogen fixation and presumably the accelerated maturation form of the nodules were observed with the use of a biofertilizer.

Structures of rhizobial lipopolysaccharides and their role in symbiosis process / Struktura lipopolisacharydów rizobiowych i ich znaczenie w procesie symbiozy.

Lipopolysaccharides synthesized by rhizobia have a various structure. Differences are observed in lipid A (considered as the most conservative part of LPS), in the core region, and in the O-specific polysaccharide. Lipids A may have different compositions of the sugar backbone and the acylation pattern. The core region of rhizobia mainly consists of hexoses, uronic acids, N-acetylquinohozamine, and Kdo, but has no heptose region typical for enterobacteria. The O-PSs may have a different structure even among strains of the same species. They are built of various monosaccharides and are often hydrophobic. An appropriate structure of LPS domains is required for establishment of an effective symbiosis between bacteria and their plant host. Changes in the structure of LPS (most often caused by mutations) resulted in a decrease in efficiency or failure of atmospheric nitrogen fixation. Complete LPS protects symbiotic bacteria penetrating plant cells and determines the proper organization and maturation of symbiosomes.

Lipid A from Oligotropha carboxidovorans Lipopolysaccharide That Contains Two Galacturonic Acid Residues in the Backbone and Malic Acid A Tertiary Acyl Substituent.

The free-living Gram-negative bacterium Oligotropha carboxidovorans (formerly: Pseudomonas carboxydovorans), isolated from wastewater, is able to live in aerobic and, facultatively, in autotrophic conditions, utilizing carbon monoxide or hydrogen as a source of energy. The structure of O. carboxidovorans lipid A, a hydrophobic part of lipopolysaccharide, was studied using NMR spectroscopy and high-resolution mass spectrometry (MALDI-ToF MS) techniques. It was demonstrated that the lipid A backbone is composed of two d-GlcpN3N residues connected by a ß-(1→6) glycosidic linkage, substituted by galacturonic acids (d-GalpA) at C-1 and C-4' positions. Both diaminosugars are symmetrically substituted by 3-hydroxy fatty acids (12:0(3-OH) and 18:0(3-OH)). Ester-linked secondary acyl residues (i.e., 18:0, and 26:0(25-OH) and a small amount of 28:0(27-OH)) are located in the distal part of lipid A. These very long-chain hydroxylated fatty acids (VLCFAs) were found to be almost totally esterified at the (ω-1)-OH position with malic acid. Similarities between the lipid A of O. carboxidovorans and Mesorhizobium loti, Rhizobium leguminosarum, Caulobacter crescentus as well as Aquifex pyrophylus were observed and discussed from the perspective of the genomic context of these bacteria.

Three-Dimensional Segmentation and Reconstruction of Neuronal Nuclei in Confocal Microscopic Images.

The detailed architectural examination of the neuronal nuclei in any brain region, using confocal microscopy, requires quantification of fluorescent signals in three-dimensional stacks of confocal images. An essential prerequisite to any quantification is the segmentation of the nuclei which are typically tightly packed in the tissue, the extreme being the hippocampal dentate gyrus (DG), in which nuclei frequently appear to overlap due to limitations in microscope resolution. Segmentation in DG is a challenging task due to the presence of a significant amount of image artifacts and densely packed nuclei. Accordingly, we established an algorithm based on continuous boundary tracing criterion aiming to reconstruct the nucleus surface and to separate the adjacent nuclei. The presented algorithm neither uses a pre-built nucleus model, nor performs image thresholding, which makes it robust against variations in image intensity and poor contrast. Further, the reconstructed surface is used to study morphology and spatial arrangement of the nuclear interior. The presented method is generally dedicated to segmentation of crowded, overlapping objects in 3D space. In particular, it allows us to study quantitatively the architecture of the neuronal nucleus using confocal-microscopic approach.

Studies on lipid A isolated from Phyllobacterium trifolii PETP02T lipopolysaccharide.

The structure of lipid A from lipopolysaccharide of Phyllobacterium trifolii PETP02T, a nitrogen-fixing symbiotic bacterium, was studied. It was found that the lipid A backbone was composed of two 2,3-diamino-2,3-dideoxy-D-glucose (GlcpN3N) residues connected by a ß-(1 â†’ 6) glycosidic linkage, substituted by galacturonic acid (GalpA) at position C-1 and partly decorated by a phosphate residue at C-4' of the non-reducing GlcpN3N. Both diaminosugars were symmetrically substituted by 3-hydroxy fatty acids (14:0(3-OH) and 16:0(3-OH)). Ester-linked secondary acyl residues [i.e. 19:0cyc and 28:0(27-OH) or 28:0(27-4:0(3-OMe))] were located in the distal part of lipid A. A high similarity between the lipid A of P. trifolii and Mesorhizobium was observed and discussed from the perspective of the genetic context of both genomes.

Structure of O-specific polysaccharide of Oligotropha carboxidovorans OM5 - a wastewater bacterium.

Oligotropha carboxidovorans strain OM5 (previously known as Pseudomonas carboxydovorans OM5) is a rod-shaped Gram-negative bacterium isolated from wastewater. This bacterium is able to live in aerobic and, facultatively, in autotrophic conditions. For autotrophic growth, the bacteria can utilize carbon monoxide or hydrogen as a source of energy. The O-specific polysaccharide isolated from O. carboxidovorans OM5 lipopolysaccharide was structurally characterized using chemical analyses, 1D and 2D NMR spectroscopy, and MALDI-TOF mass spectrometry techniques. The polysaccharide was found to be a homopolymer built up of 3-O-methyl-α-d-mannose residues linked by (1 â†’ 2)-glycosidic bonds. The degree of polymerization of high-molecular-weight polysaccharide was estimated at approximately 35-40 units. The structure of the homopolymer is depicted below: [Formula: see text].

Rhizobium strains differ considerably in outer membrane permeability and polymyxin B resistance.

Six rhizobium (Rhizobium leguminosarum bv. Trifolii TA1, Sinorhizobium meliloti 1021, Mesorhizobium huakuii IFO 15243(T), Ochrobactrum lupini LUP 21(T), Bradyrhizobium japonicum USDA110 and B. elkanii USDA 76) and two Escherichia coli strains (E. coli ATCC 25922 and E. coli HB 101) were compared in respect to polymyxin B and EDTA resistance, as well as bacterial outer membrane (OM) permeability to a fluorescent hydrophobic agent (N-phenyl-1-naphthylamine - NPN). TEM (Transmission Electron Microscopy) and a microbial test demonstrated that all the rhizobia were much more resistant to polymyxin B in comparison with E. coli strains. EDTA and polymyxin B enhance permeability of B. japonicum and O. lupini OM. Other rhizobia incorporated NPN independently of the presence of membrane-deteriorating agents; however, the level of fluorescence (measured as NPN absorption) was strain dependent.

Structure of the O-specific polysaccharide from the legume endosymbiotic bacterium Ochrobactrum cytisi strain ESC1(T).

The O-specific polysaccharide was obtained from the lipopolysaccharide of the legume-endosymbiotic bacterium Ochrobactrum cytisi strain ESC1(T) and studied by chemical analyses and 1D and 2D NMR spectroscopy. The polysaccharide was found to have a disaccharide repeating unit containing α-d-fucose and ß-N-acetyl-d-galactosamine residues connected via (1→3)-glycosidic bonds, resulting in the following structure: →3)-α-d-Fucp-(1→3)-ß-d-GalpNAc-(1→ The d-GalpNAc residue was nonstoichiometrically substituted with a 4-O-methyl group (∼10%) or with a 4,6-O-(1-carboxy)-ethylidene residue (pyruvyl group) (∼10%).

The O-specific polysaccharides from Phyllobacterium trifolii PETP02(T) LPS contain 3-C-methyl-D-rhamnose.

The O-specific polysaccharides of Phyllobacterium trifolii PETP02(T), a microsymbiont of Trifolium pratense, were obtained by mild acid hydrolysis of the lipopolysaccharide and studied by chemical analyses, mass spectrometry, and (1)H and (13)C NMR spectroscopy, including homonuclear ((1)H,(1)H DQF-COSY, TOCSY, NOESY) and heteronuclear ((1)H,(13)C HSQC, HMQC, HMBC) experiments. It was revealed that α-D-rhamnose and ß-3-C-methyl-D-rhamnose (evalose) were the only components of two identified O-polysaccharides. The major O-polysaccharide was found to consist of linear hexasaccharide repeating units, while the other minor one, is composed of disaccharide repeats. The following structures of two O-polysaccharides were established: → 2)-ß-D-Rhap3CMe-(1 → 3)-α-D-Rhap-(1 → 3)-α-D-Rhap-(1 → 2)-ß-D-Rhap3CMe-(1 → 3)-α-D-Rhap-(1 → 2)-α-D-Rhap-(1 → and → 2)-ß-D-Rhap3CMe-(1 → 3)-α-D-Rhap-(1 →.

Novel higher-order epigenetic regulation of the Bdnf gene upon seizures.

Studies in cultured cells have demonstrated the existence of higher-order epigenetic mechanisms, determining the relationship between expression of the gene and its position within the cell nucleus. It is unknown, whether such mechanisms operate in postmitotic, highly differentiated cell types, such as neurons in vivo. Accordingly, we examined whether the intranuclear positions of Bdnf and Trkb genes, encoding the major neurotrophin and its receptor respectively, change as a result of neuronal activity, and what functional consequences such movements may have. In a rat model of massive neuronal activation upon kainate-induced seizures we found that elevated neuronal expression of Bdnf is associated with its detachment from the nuclear lamina, and translocation toward the nucleus center. In contrast, the position of stably expressed Trkb remains unchanged after seizures. Our study demonstrates that activation-dependent architectural remodeling of the neuronal cell nucleus in vivo contributes to activity-dependent changes in gene expression in the brain.