Calcium as a Modulator of the Adenylyl Cyclase Activity of Potato Cells in Bacterial Pathogenesis.

This is the first study to detect the effect of calcium ions on the activity of transmembrane adenylyl cyclase (tmAC), the key enzyme of the adenylyl cyclase signaling system, under normal conditions and after a short-term exposure to exopolysaccharides (EPS) of the bacterial ring rot pathogen Clavibacter michiganensis ssp. sepedonicus (Cms). After the treatment of the roots of plants with the Cms EPS, the response to Ca2+ changed: the activity of the tmAC of plants of the resistant cultivar significantly increased, whereas in the cells of the susceptible cultivar it remained unchanged.

[Effect of N-phenyl-2-naphthylamine on activity of adenylate cyclase signal system components and virulence of bacterial phytopathogens and mutualists].

The effect of N-phenyl-2-naphthylamine, negative allelochemical isolated from the exudates of roots of pea (Pisum sativum L.), on the growth and activity of the adenylate cyclase signal system and virulence factors of the bacteria Rhizobium leguminosarum bv. viciae and Pseudomonas siringae pv. pisi was studied. It was demonstrated that N-phenyl-2-naphthylamine at a physiological concentration nonspecifically inhibited the growth of these bacteria in both planktonic cultures and biofilms. One of the reasons for this phenomenon is the reduction of intra- and extracellular concentrations of cAMP due to greater activation of phosphodiesterase, which disrupts cAMP, in comparison to soluble adenylyl cyclase, which synthesizes it. At the same time, N-phenyl-2-naphthylamine did not affect activity of either membrane-bound adenylyl cyclase or bacterial virulence factors.

Determination of cAMP in plant cells by a modified enzyme immunoassay method.

Presently, there is no doubt about the functioning of the adenylate cyclase signaling system in plants, but the role of this system in various physiological-biochemical processes has been investigated insufficiently. Cyclic adenosine monophosphate (cAMP), the key component produced by adenylate cyclase, whose concentrations in plant cells vary rather widely, is the indicator of functional activity for this signaling way. In the latter case, in the process of determination of concentrations of this messenger, one encounters difficulties related to insufficient sensitivity of the methods most frequently applied. In this connection, the proposed mechanism is a modification of the method of the enzyme immunoassay (EIA), which is based on immediate measurement of cAMP concentrations in the sample with the use of antibodies. This modification allows us to determine the concentrations of cAMP with the precision of 5 pM, which exceeds the sensitivity of other methods by approximately 10 times. The specificity of the assay has been confirmed by other two independent tests--the capillary electrophoresis and the nuclear magnetic resonance (NMR). It has also been compared to the data obtained with the use of the commercial kit from Sigma-Aldrich. The modification has been tested on such plant objects as in vitro potato plants, and suspension cells of potato and Arabidopsis.

[Isolation of bacteria of the family enterobacteriaceae from plant tissues].

Bacteria of the family Enterobacteriaceae were isolated from the tissues of a number of wild and cultivated plants. All the cultures isolated had a broad spectrum of resistance to antibiotics and were highly adhesive to human erythrocytes. The studies conducted indicate the possibility of concentration of microorganisms pathogenic for humans in plant tissues.

[A simplified method of determination of cAMP in plants using modified enzyme immunoassay].

A modification of enzyme immunoassay for quantification of cAMP and its derivatives in plants is suggested, which is based on the use of cAMP-specific primary rabbit antibodies and secondary goat antibodies conjugated with peroxidase. The lower threshold of this method is 10 pM. This technique is highly specific, simple, and inexpensive.

Properties of extracellular polysaccharides of potato ring rot pathogen and corresponding recognition sites of potato cell walls.

The properties of extracellular polysaccharides of the potato ring rot pathogen Clavibacter michiganensis subsp. sepedonicus (Cms) and the corresponding recognition sites isolated from cell walls of potato suspension cultures have been studied. Extracellular polysaccharides of Cms consist of 4-6 components, which differ greatly in molecular mass (from <1 kD to >700 kD), and are capable of formation of associates stabilized by electrostatic interactions in the presence of calcium. Using affinity column chromatography, sites possessing affinity for the total extracellular polysaccharide complex of Cms were isolated from cell walls of suspension cultures of three potato varieties with different resistance to the pathogen. The content of the receptor sites consisting of glycopeptides and sugars for the variety devoid of resistance was 10 times greater than that for the resistant variety. In the receptor fraction for the latter variety, only sugars were found. The molecular masses of the components of the receptor fraction of cell walls were from 39 to 86 kD. Polypeptides in the recognition sites for the resistant variety escaped detection in electrophoretic patterns. Study of the amino acid composition of the receptor sites of cell walls showed that the sites of the resistant variety contained trace amounts of only seven amino acids. In the sites of the receptive variety 14 amino acids were found, the content of polar amino acids being twice as large as that of nonpolar amino acids. Among polar amino acids, glutamine and glycine prevailed, whereas among nonpolar amino acids valine was dominant. We suggest that one of the reasons of variety-specific resistance of potato to Cms is the absence or a low content of the sites revealing the affinity for bacterial extracellular polysaccharides on the plant cell surface.

[Origin of invaginations and vacuoles in liposomes under the effect of endocytosis inducers including oxidants]. / Voznikhovenie invaginatsii i vezikul u liposom pod vliianiem induktorov éndotsitoza, v tom chisle oksidantov.

The action of various substances on the morphology of multilayer membranes made of lipids of egg yolk was studied. It has been shown that electroneutral and anionic compounds scaresly affected liposomes, whereas substances with pronounced basic properties, i.e. peroxidase, hemoglobin, cytochrome c, and RNAase, as well as lanthanium ions, induced the formation of invaginations, vesicles and aggregation of liposomes. Metals with variable valency: Cu2+, Cu4+, Ru6+, including lipid oxidants Fe3+ and UO+, produced similar morphological changes more intensely and moreover destroyed liposomal membranes. The activator of lipid peroxidation, namely ascorbic acid, intensified while antioxidizers such as alpha-tocopherylacetate and ionol removed the action of Fe3+ on liposomes. A protective effect was displayed by Ca2+ and Mg2+ ions and due to an increase in pH medium. Since many tested substances with the basic properties stimulate endocytosis of cells the processes of lipid peroxidation and electrostatic interactions are supposed to be part of endocytosis mechanism which does not involve the metabolic energy. It is also assumed that endocytosis may arise at the stage of protocells in terms of evolutions.